binding capacity of bull spermatozoa to
TRANSCRIPT
Aus dem Institut für Reproduktionsmedizin
der Tierärztlichen Hochschule Hannover ___________________________________________________________________
BINDING CAPACITY OF BULL SPERMATOZOA TO
OVIDUCTAL EPITHELIUM IN VITRO AND ITS RELATION TO
SPERM CHROMATIN STABILITY, SPERM VOLUME
REGULATION AND FERTILITY
INAUGURAL-DISSERTATION zur Erlangung des Grades eines
DOKTORS DER VETERINÄRMEDIZIN
(Dr. med. vet.)
durch die Tierärztliche Hochschule Hannover
Vorgelegt von
Abdel-Tawab Abdel-Razek Yassin Khalil
aus EL-FAYOUM / ÄGYPTEN
HANNOVER 2004
____________________________________________________________________
II
Scientific supervision:
Univ. Prof. Dr. rer. nat. Dr. med. habil. Edda Töpfer-Petersen
Apl. Prof. Dr. med. vet. Dagmar Waberski
Referees:
1- Apl.-Prof. Dr. med. vet. Dagmar Waberski
2- Prof. Dr. H. Bollwein
Date of the oral examination: 18.11.2004
____________________________________________________________________
III
TO MY FAMILY, especially my PARENTS
____________________________________________________________________
IV
Table of contents ____________________________________________________________________
V
TABLE OF CONTENTS
TABLE OF CONTENTS ..............................................................................................V
LIST OF FIGURES....................................................................................................XII
LIST OF TABLES.....................................................................................................XIII
LIST OF ABBREVIATIONS .................................................................................... XIV
1 INTRODUCTION ...................................................................................................... 1
2 REVIEW OF LITERATURES.................................................................................... 3
2.1 THE OVIDUCTS AND SPERM STORAGE ........................................................ 3
2.1.1 FUNCTIONAL ANATOMY OF THE OVIDUCT........................................................... 3
2.1.2 SPERM TRANSPORT IN THE FEMALE GENITAL TRACT........................................... 3
2.1.3 OVIDUCTAL SPERM RESERVOIR........................................................................ 4
2.1.3.1 Formation of the oviductal-sperm reservoir ......................................... 5
2.1.3.2 Demonstration of the sperm oviduct binding ....................................... 7
2.2 FUNCTIONS OF THE OVIDUCTAL SPERM RESERVOIR................................ 8
2.2.1 SPERM SELECTION ......................................................................................... 8
2.2.2 MAINTAINING VIABILITY, MOTILITY AND FERTILIZING CAPACITY OF SPERMATOZOA .. 9
2.2.3 REGULATION OF SPERM CAPACITATION........................................................... 10
2.2.4 REDUCTION THE INCIDENCE OF POLYSPERMY.................................................. 11
2.3 SPERM RELEASE FROM THE OVIDUCTAL RESERVOIR ............................ 12
2.4 IN VITRO SYSTEMS TO STUDY THE SPERM OVIDUCT INTERACTION ...... 13
2.4.1 TISSUE EXPLANTS ........................................................................................ 13
2.4.2 OVIDUCTAL EPITHELIAL CELLS MONOLAYERS (OECM)..................................... 13
2.5 SPERM CHROMATIN STABILITY................................................................... 14
2.5.1 SPERM CHROMATIN PACKAGING..................................................................... 14
Table of contents ____________________________________________________________________
VI
2.5.2 FACTORS AFFECTING SPERM-CHROMATIN STABILITY ........................................ 18
2.5.2.1 Age of the semen donors and aging of spermatozoa ........................ 18
2.5.2.2 Temperature...................................................................................... 18
2.5.2.3 Cryoconservation .............................................................................. 19
2.5.2.4 Reactive oxygen species (ROS)........................................................ 19
2.5.2.5 Trace elements and other factors...................................................... 20
2.5.3 EVALUATION OF SPERM CHROMATIN STABILITY ................................................ 21
2.5.3.1 Sperm chromatin structure assay (SCSA)......................................... 21
2.5.3.2 Modified fluorescence microscopical SCSA (mf-SCSA).................... 22
2.5.4 THE SPERM CHROMATIN STATUS AND FERTILITY .............................................. 23
2.5.5 EFFICIENCY OF SCSA TO PREDICT FERTILITY ................................................. 24
2.6 SPERM MEMBRANE INTEGRITY................................................................... 25
2.6.1 STRUCTURAL INTEGRITY OF SPERM MEMBRANE............................................... 25
2.6.2 FUNCTIONAL INTEGRITY OF SPERM MEMBRANE................................................ 26
2.6.2.1 The hypo-osmotic swelling test (HOST) ............................................ 26
2.6.2.2 Modified hypo-osmotic swelling test (m-HOST)................................. 27
2.6.3 EFFICIENCY OF THE HOST TO PREDICT FERTILITY........................................... 27
3 MATERIALS AND METHODS ............................................................................... 29
3.1 SEMEN SOURCE ............................................................................................ 29
3.2 HANDLING OF FROZEN-THAWED SEMEN................................................... 29
3.3 TRADITIONAL SPERMATOLOGICAL PARAMETERS .................................. 29
3.3.1 MOTILITY PARAMETERS................................................................................. 29
3.3.1.1 Subjective sperm motility................................................................... 29
3.3.1.2 Assessment of motility using cell motion analyzer (CMA) ................. 30
3.3.2 MORPHOLOGICAL ABNORMALITIES OF SPERMATOZOA ...................................... 30
3.4 ADVANCED SPERMATOLOGICAL PARAMETERS ...................................... 33
3.4.1 VIABILITY ASSESSMENT USING LIVE/DEAD®
SPERM VIABILITY KIT .................. 33
3.4.2 ASSESSMENT OF THE FUNCTIONAL SPERM MEMBRANE INTEGRITY USING MODIFIED
HYPO-OSMOTIC SWELLING TEST (M-HOST) .................................................... 34
3.4.2.1 General physical principles of measurement..................................... 35
Table of contents ____________________________________________________________________
VII
3.4.2.2 Calibration of CASY1 by latex beads ................................................ 36
3.4.2.3 Preparation of semen samples.......................................................... 36
3.4.2.4 Volumetric measurement .................................................................. 38
3.4.2.5 Analysis of derived volumetric parameters........................................ 38
3.4.3 EVALUATION OF SPERM CHROMATIN STATUS (MF-SCSA)................................. 41
3.4.3.1 Preparation of semen smears ........................................................... 41
3.4.3.2 Decondensation of sperm chromatin................................................. 41
3.4.3.3 Acid denaturation .............................................................................. 42
3.4.3.4 Staining with acridin orange (AO)...................................................... 42
3.4.3.5 Evaluation of the stained smears ...................................................... 43
3.4.4 OVIDUCTAL EXPLANT ASSAY (OEA) ............................................................... 44
3.4.4.1 Preparation of oviductal explants ...................................................... 44
3.4.4.2 Preparation of semen samples.......................................................... 46
3.4.4.3 Determination of sperm cell concentration ........................................ 46
3.4.4.4 Co-incubation of spermatozoa with oviductal explants...................... 47
3.4.4.5 Video-microscopy and image analysis .............................................. 47
3.4.4.6 Estimation the surface area of the explant ........................................ 48
3.4.4.7 Determination of the binding index (BI) ............................................. 48
3.4.5 IN VITRO FERTILIZATION (IVF)........................................................................ 48
3.4.5.1 Collection of ovaries .......................................................................... 49
3.4.5.2 Recovery of oocytes.......................................................................... 50
3.4.5.3 In vitro maturation (IVM).................................................................... 51
3.4.5.4 In vitro fertilization (IVF) .................................................................... 52
3.4.5.5 Preparation of spermatozoa and fertilization ..................................... 52
3.4.5.6 Removal of cumulus cells.................................................................. 53
3.4.5.7 In vitro culture of embryos (IVC)........................................................ 53
3.5 STASTICAL ANALYSIS .................................................................................. 55
3.5.1 ANALYSIS OF VOLUMETRIC PARAMETERS ........................................................ 55
3.5.2 ANALYSIS OF THE DATA OF MF-SCSA ............................................................ 55
3.5.3 ANALYSIS OF THE DATA OF OVIDUCTAL EXPLANTS ASSAY (OEA)....................... 55
3.5.4 ANALYSIS OF THE IVF DATA........................................................................... 56
Table of contents ____________________________________________________________________
VIII
3.5.5 SIGNIFICANCE LEVELS FOR THE PROBABILITY OF MISTAKE ................................ 56
4 RESULTS............................................................................................................... 57
4.1 STANDARD SPERMATOLOGICAL PARAMETERS ...................................... 57
4.1.1 MOTILITY PARAMETERS................................................................................. 57
4.1.1.1 Post-thawing subjective motility......................................................... 57
4.1.1.2 Post thawing motility using cell motion analyser (CMA) .................... 58
4.1.1.3 Motility of percoll selected spermatozoa............................................ 59
4.1.2 MORPHOLOGICAL ABNORMALITIES OF SPERMATOZOA ...................................... 61
4.1.3 RELATIONSHIP BETWEEN IVF RESULTS AND STANDARD SPERMATOLOGICAL
PARAMETERS ............................................................................................... 62
4.2 ADVANCED SPERMATOLOGICAL PARAMETERS ...................................... 64
4.2.1 VIABILITY ASSESSMENT USING LIVE/DEAD® SPERM VIABILITY KIT .................. 64
4.2.2 MODIFIED HYPO-OSMOTIC SWELLING TEST (M-HOST) ..................................... 64
4.2.2.1 Relative volume shift (RVS)............................................................... 66
4.2.2.2 Regulatory volume decrease (RVD).................................................. 67
4.2.3 MODIFIED FLORESCENCE MICROSCOPICAL SCSA (MF-SCSA) ......................... 68
4.2.3.1 Relationship between sperm chromatin status and IVF results......... 68
4.3 OVIDUCTAL EXPLANT ASSAY (OEA) ........................................................... 71
4.3.1 DIFFERENCES AMONG INDIVIDUAL BULLS ........................................................ 72
4.3.2 RELATIONSHIP BETWEEN SPERM-OVIDUCT BINDING ABILITY AND IVF RESULTS ... 73
4.4 CORRELATION MATRIX AMONG SPERMATOLOGICAL PARAMETERS ... 75
4.4.1 RELATIONSHIP BETWEEN SPERM-OVIDUCT BINDING ABILITY (BI) AND OTHER
SPERMATOLOGICAL PARAMETERS .................................................................. 76
1) Traditional spermatological parameters .................................................... 76
2) Sperm membrane functional activity (m-HOST)........................................ 76
3) Percentage of spermatozoa with unstable chromatin (mf-SCSA) ............. 76
4.4.2 RELATIONSHIP BETWEEN SPERM CHROMATIN STATUS (MF-SCSA) AND OTHER
SPERMATOLOGICAL PARAMETERS .................................................................. 76
1) Conventional spermatological parameters ................................................ 76
2) Sperm membrane functional activity (m-HOST)........................................ 76
Table of contents ____________________________________________________________________
IX
4.4.3 RELATIONSHIP BETWEEN SPERM MEMBRANE FUNCTIONAL STATUS (M-HOST) AND
STANDARD SPERMATOLOGICAL PARAMETERS.................................................. 77
5 DISCUSSION ......................................................................................................... 79
5.1 OVIDUCT EXPLANT ASSAY (OEA)................................................................ 79
5.1.1 RELATIONSHIP BETWEEN SPERM-OVIDUCTAL EPITHELIUM BINDING CAPACITY AND
CHROMATIN STABILITY .................................................................................. 81
5.1.2 RELATIONSHIP BETWEEN SPERM-OVIDUCTAL EPITHELIUM BINDING CAPACITY AND
SPERM VOLUMETRIC PARAMETERS ................................................................. 83
5.1.3 RELATIONSHIP BETWEEN SPERM OVIDUCT BINDING CAPACITY AND FERTILITY IN
VITRO .......................................................................................................... 84
5.1.4 RELATIONSHIP BETWEEN SPERM–OVIDUCT BINDING ABILITY AND STANDARD
SPERMATOLOGICAL PARAMETERS .................................................................. 85
5.2 SPERM CHROMATIN STATUS (MF-SCSA) .................................................... 86
5.2.1 RELATIONSHIP BETWEEN SPERM CHROMATIN STATUS (MF-SCSA) AND SPERM
VOLUMETRIC PARAMETERS (M-HOST) ........................................................... 87
5.2.2 RELATIONSHIP BETWEEN SPERM CHROMATIN STATUS AND IN VITRO FERTILITY ... 89
5.3 SPERM VOLUMETRIC PARAMETERS (M-HOST).......................................... 90
5.3.1 RELATIONSHIP BETWEEN SPERM VOLUMETRIC PARAMETERS (M-HOST) AND
VIABILITY OF SPERMATOZOA (SYBR14/PI) ..................................................... 92
5.3.2 RELATIONSHIP BETWEEN VOLUMETRIC PARAMETERS (M-HOST) AND FERTILITY
(IVF)........................................................................................................... 93
5.4 CONCLUSION ................................................................................................. 94
6 SUMMARY ............................................................................................................. 95
7 ZUSAMMENFASSUNG.......................................................................................... 97
8 REFERENCES ....................................................................................................... 99
9 APPENDIX ........................................................................................................... 149
Table of contents ____________________________________________________________________
X
9.1 LABORATORY NEEDS................................................................................. 149
9.2 CHEMICALS AND REAGENTS..................................................................... 150
9.3 BUFFERS, SOLUTIONS, MEDIA AND DILUENTS ....................................... 151
9.3.1 NATRIUM CHLORIDE SOLUTION 10 % (FOR KILLING OF SPERMATOZOA) ............ 151
9.3.2 FORMOLCITRAT (FOR FIXATION OF SPERMATOZOA) ........................................ 151
9.3.3 HEPES BUFFER SALINE (HBS) FOR M-HOST ................................................ 151
9.3.4 PERCOLL® SOLUTION (AFTER HARRISON ET AL., 1993) ............................. 151
9.3.5 HEPES. 0.1 % BSA-BUFFER FOR SUPRA VITAL STAINING ............................... 153
9.3.6 MATERIALS FOR MF-SCSA.......................................................................... 153
9.3.6.1 Natrium citrate buffer 2.9 % (1000 ml)............................................. 153
9.3.6.2 Carnoy’s solution, pH 2 (60 ml) ....................................................... 153
9.3.6.3 Citric acid-stock solution 0.1 molar (1000 ml).................................. 154
9.3.6.4 Di-Natriumhydrogenphosphat, 0.3 molar solution (100 ml) ............. 154
9.3.6.5 Acridin Orange (AO) stock solution (100 ml) ................................... 154
9.4 MATERIALS FOR THE OVIDUKT-EXPLANT-ASSAY .................................. 155
9.4.1 EQUIPMENTS AND INSTRUMENTS.................................................................. 155
9.4.2 PBS MEDIUM (LEFEBVRE AND SUAREZ, 1996) ....................................... 156
9.4.3 TALP MEDIUM (PARRISH ET AL. 1988)...................................................... 156
9.5 MATERIALS FOR IVF.................................................................................... 157
9.5.1 PBS STOCK SOLUTION (1000 ML) ................................................................ 157
9.5.2 SLICE MEDIUM (500 ML) .............................................................................. 157
9.5.3 TCM-AIR-MEDIUM (100 ML) ........................................................................ 157
9.5.4 TCM-PURE MEDIUM + BSA (WASH DROPS) ................................................. 157
9.5.5 TCM-PURE MEDIUM+BSA+SUIGONAN® (MATURATION DROPS) ..................... 158
9.5.6 SPERM-TALP STOCK SOLUTION (500 ML) ..................................................... 158
9.5.7 FERT-TALP STOCK SOLUTION (500 ML)........................................................ 158
9.5.8 HHE STOCK SOLUTION (HEPARIN / HYPOTAURIN / EPINEPHRINE)................... 159
9.5.8.1 250 µM Epinephrine (50 ml) ............................................................ 159
9.5.8.2 Hypotaurin 1 mM (10 ml) ................................................................. 159
9.5.8.3 Heparin 50 IE (10 ml) ...................................................................... 159
9.5.8.4 Working medium-Stock solution (40 ml).......................................... 159
Table of contents ____________________________________________________________________
XI
9.5.9 SOF MEDIUM............................................................................................. 160
9.5.9.1 SOF / Stock A-Medium (100 ml)...................................................... 160
9.5.9.2 SOF / Stock B-Medium (1 ml).......................................................... 161
9.5.9.3 SOF / Stock C-Medium (1 ml) ......................................................... 161
9.5.9.4 SOF / Stock D-Medium (1 ml) ......................................................... 161
9.5.9.5 SOF / Stock E-Medium (100 ml)...................................................... 161
10 STATUTORY DECLARATION........................................................................... 162
11 ACKNOWLEDGEMENTS .................................................................................. 163
List of Figures ___________________________________________________________________
XII
LIST OF FIGURES
FIGURE 1: EQUIVALENT LEVELS OF DNA PACKAGING IN SOMATIC AND SPERM CELLS. ........ 16
FIGURE 2: FORMS OF MORPHOLOGICALLY ABNORMAL BULL SPERMATOZOA ...................... 32
FIGURE 3: CASY1 (CELL COUNTER AND ANALYZER SYSTEM), MODEL TTC..................... 35
FIGURE 4: WASHING OF FROZEN-THAWED BULL SEMEN THROUGH A DISCONTINUOUS
PERCOLL GRADIENT. .................................................................................... 37
FIGURE 5: VOLUME DISTRIBUTION CURVES OF FROZEN-THAWED BULL SPERM UNDER ISO-
OSMOTIC (A) AND HYPO-OSMOTIC (B) CONDITIONS.......................................... 40
FIGURE 6: COMPUTER-ASSISTED EVALUATION OF SPERM CHROMATIN STATUS WITH
ANALYSIS® 3.0 PROGRAM, A) MF-SCSA ...................................................... 44
FIGURE 7: A) BOVINE SPERM BOUND TO OVIDUCTAL EPITHELIAL (PHASE CONTRAST
MICROSCOPE 200X); B) SCANNING MICROGRAPH (5000X). ............................. 45
FIGURE 8: IN VITRO BOVINE CUMULUS OOCYTE COMPLEX, CLASSES I AND II...................... 51
FIGURE 9: DIFFERENT EMBRYONIC STAGES AFTER LINDNER AND WRIGHT (1983):....... 54
FIGURE 10: POST-THAWING SUBJECTIVE PROGRESSIVE FORWARD MOTILITY %. ............... 57
FIGURE 11: POST-THAWING PROGRESSIVE FORWARD MOTILITY %, ESTIMATED WITH CMA.58
FIGURE 12: POST-THAWING SUBJECTIVE PROGRESSIVE FORWARD MOTILITY % OF PERCOLL
SELECTED SPERMATOZOA. ........................................................................... 59
FIGURE 13: TOTAL PERCENTAGE OF MORPHOLOGICALLY ALTERED SPERMATOZOA (MAS) . 62
FIGURE 14: ALIVE SPERM % AS DETERMINED USING LIVE/DEAD® SPERM VIABILITY KIT .. 64
FIGURE 15: RELATIVE SHIFT OF MODAL SPERM CELL VOLUME ......................................... 66
FIGURE 16: REGULATORY VOLUME DECREASE (RVD) ................................................... 67
FIGURE 17: PERCENTAGE OF SPERMATOZOA WITH UNSTABLE CHROMATIN....................... 68
FIGURE 18: CLEAVAGE- AND BLASTOCYST RATES IN TWO GROUPS OF BULLS. ................... 71
FIGURE 19: SPERM-OVIDUCT EXPLANTS BINDING INDICES OF INDIVIDUAL BULLS.............. 72
FIGURE 20: CLEAVAGE AND BLASTOCYST RATES IN TWO GROUPS OF BULLS WITH DIFFERENT
SPERM- OVIDUCT EXPLANT BINDING INDICES .................................................. 75
List of Tables ___________________________________________________________________
XIII
LIST OF TABLES
TABLE 1: MEAN, MINIMUM AND MAXIMUM VALUES OF SPERM MOTILITY PARAMETERS.......... 60
TABLE 2: MORPHOLOGICAL PARAMETERS AND VIABILITY OF FROZEN THAWED SEMEN (90
EJACULATES FROM 30 BULLS; 3 EJACULATES PER BULL). ................................ 61
TABLE 3: PEARSON’S CORRELATION COEFFICIENTS AND LEVELS OF SIGNIFICANCE BETWEEN
IVF RESULTS AND SOME SPERMATOLOGICAL PARAMETERS (N = 11 EJACULATE
FROM 11 BULLS). ........................................................................................ 63
TABLE 4: SPERM CELL VOLUMETRIC PARAMETERS (90 EJACULATES FROM 30 BULLS). ....... 65
TABLE 5: THE SPERM CHROMATIN STABILITY % AND OTHER SPERMATOLOGICAL PARAMETERS
FOR TWO GROUPS OF BULLS (VALUES ARE AVERAGES OF 3 EJACULATES PER BULL
.................................................................................................................. 69
TABLE 6: CLEAVAGE- AND BLASTOCYST RATES OF INDIVIDUAL BULLS IN RELATION TO THEIR
CHROMATIN INSTABILITY % (N = 5 BULLS). ..................................................... 70
TABLE 7: CLEAVAGE AND BLASTOCYST RATES OF TWO GROUPS BULLS WITH LOW AND HIGH
BINDING INDICES (N= 6 BULLS)...................................................................... 73
TABLE 8: SPERM OVIDUCT BI AND SPERMATOLOGICAL PARAMETERS OF TWO GROUPS OF
BULLS (N = 6). VALUES WITH DIFFERENT LETTERS IN THE SAME ROW ARE
SIGNIFICANTLY DIFFERENT............................................................................ 74
List of abbreviations ___________________________________________________________________
XIV
LIST OF ABBREVIATIONS
AI AO BI BOEC BR BSA °C ca Ca2+
CASY1 Cm CMA CR CTC DMSO DNA DTT D.W eCG EDTA EN et al Explant Fig g h HCG Hepes HHE HOST IU IVC IVF IVM IVP Kg L M mf-SCSA mg µg µl µm min m-HOST Mio
Artificial insemination Acridin orange Binding index Bovine oviductal epithelial cells Blastocyst rate Bovine serum albumin Grade Celsius Circa Calcium ion Cell counter and analyzer system Centimetre Cell motion analyzer Cleavage rate Chlortetracycline Dimethylsulfoxide Deoxyribonucleic acid 1,4 Dithiotreit Double distilled water Equine chorionic gonadotropin Ethylene di-amine tetra acetic acid Eosin/Nigrosin et alii (and others) Part or section of living tissue which taken out from the natural site of growth and place in a medium for culture Figure Gram Hour Human chronic gonadotropine H-[2-Hydroxyethyl] piperazin-N’-[Ethansulfonic acid] Heparin, hypotaurin and epinephrin Hypo-osmotic swelling test International unit In vitro culture In Vitro Fertilization In vitro maturation In vitro embryo production Kilogram Litre Molar Modified fluorescence microscopical sperm chromatin structure assay Milligram Microgram Micro litre Micrometer Minute Modified hypo-osmotic-swelling Test Million
List of abbreviations ___________________________________________________________________
XV
ml mM mOsm/kg mOsm/L No OEA OECE OECM p PBS PH PI PVA PVP ® RVD RVS SCSA SD sec SEM SOF TALP TB TCM TM UTJ VAP VCL VSL Vi5m Vh5m Vi20m Vh20m RVSm Vr20m RVDm Vi5 Vh5 Vi20 Vh20 Vr20 RVS RVD x
Millilitre Millimolar Milliosmolal Milliosmolar Number Oviduct-explant-assay Oviduct epithelial cells explants Oviduct epithelial cell monolayers Probability of the zero hypotheses Phosphate buffered saline Hydrogen ion concentration Propidium iodide Polyvinyl alcohol Polyvinyl pyrrolidone Registered trade mark Regulative volume decrease of modal sperm volume Relative volume shift (modal value) Sperm chromatin structure assay Standard deviation Second Standard error of the mean Synthetic oviduct fluid Tyrode, Albumin, Lactate, Pyruvate medium Trypan blue Tissue culture medium Total motile spermatozoa Utero-tubal junction Average path velocity of spermatozoa (µm/sec.) Curvilinear velocity of spermatozoa (µm/sec.) Straight-line velocity of spermatozoa (µm/s) Mean sperm volume under iso-osmotic conditions at 5min Mean sperm volume under hypo-osmotic condition at 5 min Mean sperm volume under iso-osmotic condition at 20 min Mean sperm volume under hypo-osmotic condition at 20 min Relative shift of mean sperm volume after 5 min Relative shift of mean sperm volume after 20 min Regulative decrease of mean sperm volume Modal sperm volume under iso-osmotic conditions at 5min Modal sperm volume under hypo-osmotic condition at 5 min Modal sperm volume under iso-osmotic condition at 20 min Modal sperm volume under hypo-osmotic condition at 20 min Relative shift of modal sperm volume after 20 min Relative volume shift of modal values after 5 min Regulative decrease of modal sperm volume Arithmetic mean
___________________________________________________________________
XVI
Introduction ___________________________________________________________________
1
1 INTRODUCTION
In vivo several physiological barriers exist to ensure that spermatozoa that participate
in fertilization are of the highest quality. Such barriers are thought to include
penetration of the cervical mucus, transport through the female reproductive tract,
storage and survival in the oviduct, penetration of cumulus cells and binding to the
zona pellucida (HUNTER, 1995; BAILLIE et al., 1997). Specifically, sperm that reach
the fallopian tube have a higher proportion of normal morphology than that seen for
sperm lower in the tract (MORTIMER et al., 1982). At the time of insemination in
many eutherian mammals millions of sperm are released to the reproductive tract,
only thousands pass through the uterotubal junction into the oviductal isthmus, where
they form a reservoir (HUNTER, 1998; LEFEBVRE et al., 1995). The sperm reservoir
is created in various mammalian species such as cattle (HUNTER and WILMUT,
1984), rabbit (OVERSTREET and COOPER, 1978b), sheep (HUNTER and NICHOL,
1983), pigs (HUNTER, 1981), hamster (SMITH et al., 1987), and mice (SUAREZ,
1987). The sperm reservoir acts to ensure that enough fertile spermatozoa are
available in the oviduct when ovulation occurs. Contact of the spermatozoa with the
oviduct epithelial cells maintains viability of spermatozoa stored in the oviduct
(SMITH and YANAGIMACHI, 1990) and prolongs their ability to fertilize (POLLARD
et al., 1991; SMITH and YANAGIMACHI, 1991). As the time of ovulation nears,
release of spermatozoa commences, and a few reaches the ampulla where
fertilization takes place (HUNTER, 1998; SUAREZ et al., 1990). The interaction takes
place between the sperm plasma membrane overlying the acrosome and the surface
of the mucosal epithelium often via cilia (LEFEBVRE et al. 1995; SUAREZ, 1987;
HUNTER et al., 1991; SMITH and YANAGIMACHI, 1991).
In the recent years, the interaction of spermatozoa with the oviduct epithelium has
been studied using in vitro culture of spermatozoa and epithelial cells and more
focused on the mechanisms and biological aspects of sperm oviduct binding. The
following in vitro studies were conducted to further characterize functional aspects of
the interaction between frozen-thawed bull spermatozoa and the oviductal
epithelium. Differences between the initial binding capacity of frozen-thawed sperm to
Introduction ___________________________________________________________________
2
oviductal epithelium using the oviductal explant assay (OEA) were studied in
repeated ejaculates from 30 bulls. Additionally, the relationship between sperm-
oviductal epithelium binding capacity and membrane functional integrity and
chromatin stability, and also their relation to fertility in vitro were investigated.
Review of literatures ___________________________________________________________________
3
2 REVIEW OF LITERATURES
2.1 THE OVIDUCTS AND SPERM STORAGE
2.1.1 Functional anatomy of the oviduct
The mammalian oviducts are anatomically divided into three sections, which in a
caudal to cranial direction are: the isthmus, the ampulla and the infundibulum. The
distinct cell types of the epithelium lining the oviductal isthmus are ciliated epithelial
cells, which presumably facilitate gamete movement within the oviduct, and non-
ciliated secretory cells, which are characterized by secretory granules and by
microvilli on their apical surface. The morphology and ultrastructure of ciliated and
secretory oviductal epithelial cells (OEC) have been described in large domestic
animal species (cattle: STALHEIM et al., 1975; ABE and OIKAWA, 1993; BOLLO et
al., 1990; goats: ABE et al., 1993; pigs: WU et al., 1976; horses: BOYLE et al., 1987;
BADER, 1982; BALL, 1996) and changes in cell structure associated with the cycle
stage of the female have been documented (ABE and OIKAWA, 1993; ABE et al.,
1993). In the primate oviducts, cycles of ciliation and deciliation of the fimbria and
ampulla have been reported, and reciliation appears to be controlled by circulating
estrogen concentrations (BRENNER et al., 1974; ODOR et al., 1980). The epithelial
height and activity of the secretory cells are increased around the time of ovulation
(JANSEN, 1984; AMSO et al., 1994). The energy substrates as glucose, pyruvate
and lactate originate from the plasma transudate through passive diffusion and the
cells show glycolitic activity (LEESE, 1988; EDWARDS and LEESE, 1993), resulting
in a low glucose concentration and some accumulation of lactate in the luminal fluid.
2.1.2 Sperm transport in the female genital tract
Sperm migration through the female tract is sustained by sperm swimming activity,
muscular contraction of the tract and ciliary activity of the oviductal mucosa
(BENNETT et al., 1988; AYAD at al., 1990; BARRATT and COOKE, 1991).
Depending on the species and on the site of sperm deposition, by 1-8 hours after
Review of literatures ___________________________________________________________________
4
insemination the fertilizing sperm will be found stored in the caudal isthmus. However
uterine sperm may continue to enter the oviductal isthmus. Therefore, sperm cells
may remain in the isthmus as long as 18-20 h in cattle, 17-18 h in sheep, and up to
36 h in pigs. Since most large farm animals will be mated at a specific phase of the
estrous cycle, spermatozoa are present in the reproductive tract at or around the time
of ovulation, establishing a temporal relationship between semen deposition and
ovulation (HUNTER 1984; ORIHUELA et al., 1999). Moreover it was mentioned that
sperm transport in to the oviducts involving adovarian waves of contraction and
ciliary’s activity in the oviductal epithelium in rabbit (VANDEN-BOSCH and HAFEZ,
1974), pigs (BLANDAU and GADDUM-ROSSE, 1974), cattle (WILMUT and
HUNTER, 1984), sheep (HUNTER and NICHOL, 1983) and humans (AHLGREN,
1975; BARRATT and COOKE, 1991). In horses the fertilizing population of
spermatozoa reached the oviduct within two to four hours of insemination (BRINSKO
et al., 1990; 1991), whereas this transport might take about eight hours in cattle
(HAWK, 1987). Recently, an association between impaired sperm transport and
subfertility has been suggested in horses (SCOTT et al., 1995).
2.1.3 Oviductal sperm reservoir
After natural mating, sperm are transported to the oviduct, where they form a
reservoir. In species where sperm are deposited in the vagina, sperm enter the cervix
and are stored in crypts from which they are subsequently released to reach the
oviduct (HARPER, 1994). Evidence from studies in a variety of mammalian species
supports the existence of sperm storage in the female oviduct prior to fertilization. In
vivo studies characterizing oviductal sperm storage have been reported in mice
(ZAMBONI, 1972; SUAREZ, 1987), rats (SHALGI and KRAICER, 1978), guinea pigs
(YANAGIMACHI and MAHI, 1976), hamster (SMITH et al., 1987; SMITH and
YANAGIMACHI, 1990; 1991), rabbits (OVERSTREET et al., 1978), sheep (HUNTER
et al., 1980; 1982; HUNTER and NICHOL, 1983) pigs (HUNTER, 1981; HUNTER et
al., 1987), cattle (HUNTER and WILMUT, 1984; LARSSON and LARSSON, 1986;
HUNTER et al., 1991; LEFEBVRE et al., 1995), horses (PARKER et al., 1975;
BADER, 1982), bats (RACY, 1975), marsupials (TAGGART and TEMPEL-
Review of literatures ___________________________________________________________________
5
SMITH,1991; BEDFORD and BREED,1994) and humans (AHLGREN,1975;
MANSOUR et al.,1993; HUNTER, 1995). Evidence for oviductal sperm storage has
also been derived from studies in vitro (cattle: ELLINGTON et al., 1991; POLLARD et
al., 1991; Pigs: SUAREZ et al., 1991; horses: LEFEBVRE and SAMPER, 1993;
ELLINGTON et al., 1993a; THOMAS et al., 1994a,b; humans: BONGSO et al., 1993;
PACEY et al., 1995; MORALES et al., 1996). Prolonged storage of sperm in the
female reproductive tract is also a well-described phenomenon in poultry (BAKST,
1994). In contrast, WILLIAMS et al. (1993) did not found evidence for an isthmic
oviductal sperm reservoir in humans. It is noteworthy that while an oviductal sperm
reservoir exists in marsupial mammals, the sperm do not appear to attach to the OEC
(BEDFORD and BREED, 1994).
2.1.3.1 Formation of the oviductal-sperm reservoir
Several mechanisms have been proposed for the retention of spermatozoa in the
oviductal isthmus. Spermatozoa could remain in the isthmus due to depressed sperm
motility, as reduction of flagellar beat activity in spermatozoa in contact with the OEC
has been reported (OVERSTREET and KATZ, 1977; SUAREZ, 1987; SUAREZ and
OSMAN, 1987). Alternatively, it has been suggested that spermatozoa retained in the
isthmic reservoir may be due to: localized constriction of the isthmus (OVERSTREET
and COOPER, 1975; SUAREZ, 1987), attachment to the OEC (POLLARD et al.,
1991; HUNTER, 1991), entrapment in locally secreted mucus (SUAREZ, 1987;
SUAREZ et al., 1991), estrous stage related edema of oviductal mucosa (FLECHON
and HUNTER, 1981; BOYLE et al., 1987) or a combination of these factors.
Scanning electron microscopy of oviductal tissue recovered from bred mares and
cows revealed spermatozoa in intimate contact with OEC (BADER, 1982; BOYLE et
al., 1987; LEFEBVRE et al., 1995), and this was confirmed by studies using
cocultures of spermatozoa and OEC from horses and cattle (ELLINGTON et al.,
1991; 1993a; THOMAS et al., 1994a,b; SUZUKI and FOOTE, 1995). Some authors
reported that spermatozoa attaching only to ciliated OEC (sheep: HUNTER and
NICHOL, 1983; pigs: SUAREZ et al., 1991; PETRUNKINA et al., 2001b; cattle:
POLLARD et al., 1991; LEFEBVRE et al., 1995; horses: ELLINGTON et al., 1993b),
Review of literatures ___________________________________________________________________
6
whereas other studies revealed spermatozoa attaching to both ciliated and non-
ciliated cells (hamsters: SMITH and YANAGIMACHI, 1990; cattle: HUNTER et al.,
1991; SUZUKI and FOOTE, 1995; horses: BADER, 1982; THOMAS et al., 1994a). It
is noteworthy that when bovine sperm placed into culture of fetal OEC bound only to
the differentiated OEC that possessed cilia (POLLARD, 1992). On the other hand,
sperm can bind to OEC monolayers that lack cilia (THOMAS et al., 1994b;
GUTIERREZ et al., 1993). The storage of spermatozoa in oviductal reservoir appears
to be physical attachment of spermatozoa to the OEC (mouse: SUAREZ, 1987;
hamster: SMITH and YANAGIMACHI, 1989; 1990; rabbit: OVERSTREET and
COOPER, 1978a,b; pig: HUNTER, 1981; 1984; FLECHON and HUNTER, 1981;
SUAREZ et al., 1991; cattle: LARSSON and LARSSON, 1985; HUNTER et al., 1991;
LEFEBVRE et al., 1995; horses: BADER, 1982). The interaction takes place between
the sperm plasma membrane overlying the acrosome and the surface of the mucosal
epithelium often via cilia (SUAREZ, 1987; SMITH and YANAGIMACHI, 1991;
HUNTER et al., 1991; LEFEBVRE et al., 1995). Within the isthmic reservoir,
spermatozoa attach to OEC by their rostral plasma membrane (HUNTER et al., 1987,
1991; POLLARD et al., 1991; SMITH and YANAGIMACHI, 1991; SUAREZ et al.,
1991; ELLINGTON et al., 1993b; LEFEBVRE et al., 1995). Although the in vivo
research in several species indicates that sperm reservoir is limited to the caudal
isthmus (HUNTER, 1981; HUNTER and NICHOL, 1983; HUNTER, 1984; HUNTER
and WILMUT, 1984; SMITH and YANAGIMACHI, 1990; HUNTER et al., 1991), the
sperm binding with OEC in vitro is not limited to isthmic epithelium when sperm are
given equal access to isthmic and ampullary epithelial cells. No anatomical regional
effect on sperm binding in vitro was detected in pigs (SUAREZ et al., 1991;
PETRUNKINA et al., 2001b) and cattle (NAGAI and MOOR, 1990; LEFEBVRE et al.,
1995). However, in other studies an anatomical effect was observed in equine
(THOMAS et al., 1994a) and human (BAILLIE et al., 1997). Comparatively little is
known about the mechanisms of sperm binding to the OEC. Cell surface
carbohydrates are important in cell adhesion (GAHMBERG et al., 1992) and play an
important role in mammalian fertilization (AHUJA, 1985). Glycoprotein recognition
has been implicated in the adhesion of spermatozoa to OEC. Glycoproteins
Review of literatures ___________________________________________________________________
7
expressed on the luminal surface of OEC were characterized by lectin
immunocytochemistry in mice (WHYTE et al., 1987), rabbits (MENGHI et al., 1985;
1995), pigs (RAYCHOUDHURY et al., 1993), horses (BALL et al., 1997) and humans
(SCHULTE et al., 1985; WU et al., 1993). A lectin-like interaction between
spermatozoa and OEC has been demonstrated, involving recognition of feutin and
sialic acid in hamsters (DE MOTT et al., 1995), galactose in horses, (LEFEBVRE,
1997), fucose in cattle (LEFEBVRE and SUAREZ, 1996) and mannose in pigs
(WAGNER et al., 2002). It has been reported that fucose blocks binding of bull sperm
to bovine OEC in vitro (LEFEBVRE et al., 1997). Fucose-specific lectins have been
used to demonstrate that fucose is densely distributed on the surface of bovine
oviductal epithelium; furthermore, pre-treatment of OEC with fucosidase reduces
sperm binding (LEFEBVRE et al., 1997). Specific recognition and binding between
bovine sperm and homologous OEC are believed to be mediated by the interaction of
a Ca2+-dependant Lectins on the sperm head surface (DE MOTT et al., 1995;
SUAREZ et al., 1998) and Fucose supported by a Lewis-A-trisaccharide present on
the apical membrane of OEC (SUAREZ et al., 1998; LEFEBVRE et al., 1997; REVAH
et al., 2000). Similar binding of bovine sperm to OEC has also been observed in vitro
(ELLINGTON et al., 1991; POLLARD et al., 1991; CHIAN and SIRARD, 1995;
SUZUKI and FOOTE, 1995; LEFEBVRE and SUAREZ, 1996; SUZUKI et al., 1997).
This is evidence strongly suggests that fucose is involved in bovine sperm binding to
oviductal epithelium.
2.1.3.2 Demonstration of the sperm oviduct binding
Sperm binding to OEC has been observed in all eutherian mammals in which the
reservoir has been examined. Attachment of sperm to OEC has been demonstrated
in vivo by Transillumination of excised whole oviduct in mice (SUAREZ, 1987) and
hamster (KATZ and YANAGIMACHI, 1980). Scanning electron micrographs (SEM) of
oviducts of mated pigs and cows indicate an apparent association between sperm
and oviductal epithelium (SUAREZ et al., 1991; HUNTER at al., 1987; LEFEBVRE et
al., 1995). In vitro sperm binding to oviductal explants has been used as a model for
studying the sperm-oviduct interaction in several species including cattle
Review of literatures ___________________________________________________________________
8
(ELLINGTON et al., 1991; POLLARD et al., 1991; LEFEBVRE et al., 1995), pigs
(SUAREZ et al., 1991; RAYCHOUDHURY and SUAREZ, 1991), horses (LEFEBVRE
and SAMPER, 1993; ELLINGTON et al., 1993a; THOMAS et al., 1994a,b) and
humans (BONGSO et al., 1993; PACEY et al., 1995; MORALES et al., 1996). Sperm
attachment to OEC monolayers has also been demonstrated in these species
(sheep: GUTIERREZ et al., 1993; equine: ELLINGTON et al., 1993b; bovine:
GUALTIERI and TALEVI, 2003).
2.2 FUNCTIONS OF THE OVIDUCTAL SPERM RESERVOIR
2.2.1 Sperm selection
In vivo several physiological barriers exist to ensure that sperm, which participate in
fertilization, are of the highest quality. Such barriers are though to include penetration
of the cervical mucus, transport through the female reproductive tract, utero-tubal
junction (UTJ), storage in the fallopian tube, binding to and penetrate of cumulus cells
and the zona pellucida (HUNTER, 1995). Specifically, sperm that reach the fallopian
tube have a higher proportion of normal morphology than that seen for sperm lower
in the tract (MORTIMER et al., 1982), and sperm that bind to the zona pellucida have
more normal chromatin structure than those that do not (HOSHI et al., 1996). In
addition, co-culture of sperm with oviduct epithelial cells results in a stabilizing effect
for sperm against chromatin changes (ELLINGTON et al., 1998a). Establishment of
the oviductal sperm reservoir can serve to selectively retain uncapacitated,
morphologically normal sperm with intact acrosome from the population of
spermatozoa reaching the oviduct (MITCHELL et al., 1985; SHALGI et al., 1992;
THOMAS et al., 1994b; LEFEBVRE et al., 1995; PETRUNKINA et al., 2001b).
Furthermore, it was estimated that binding to oviductal cells is not only beneficial for
sperm survival but also represents a crucial step for the selection of spermatozoa
endowed with superior fertilization competence (GUALTIERI and TALEVI, 2003).
Sperm that did not attach to OEC during in vitro culture were the poorer quality sperm
in the sample. In particular, human sperm that did not attach had poorer motility and
lesser quality chromatin (ELLINGTON et al., 1999a). Recent studies suggest that an
Review of literatures ___________________________________________________________________
9
intricate cooperation and synchrony exists between mammalian sperm and both OEC
and oviductal secretion in the regulation of the events of reproduction including
sperm survival and storage at the internal body temperature, sperm selection,
capacitation and optimization of subsequent fertilization (BARRATT and COOKE,
1991; ZHU et al., 1994; KIM et al., 1996). Capacitated spermatozoa do not appear to
attach to OEC (hamster: SMITH and YANAGIMACHI, 1990; 1991; stallion: THOMAS
et al., 1995a; bull: LEFEBVRE and SUAREZ, 1996).
2.2.2 Maintaining viability, motility and fertilizing capacity of
spermatozoa
Extended lifespan of spermatozoa and maintenance of sperm motility is a widely
documented effect of sperm-oviduct interaction. In vitro incubation of spermatozoa
with oviductal fluid resulted in maintenance of sperm motility (humans: ZHU et al.,
1994; EVREV et al., 1994; cattle: MC NUTT et al., 1994; GRIPPO et al., 1995),
presumably by providing glycolyzable substrates (ENGLE at al., 1975). A beneficial
effect on sperm motility has been demonstrated using OEC conditioned medium
(ANDERSON and KILLIAN, 1994; IJAZ et al., 1994; ABE et al., 1995a;b) and
cocultures of spermatozoa with OEC (SUAREZ et al., 1991; GUERIN et al., 1991;
BASTIAS et al., 1993; GUTIERREZ et al., 1993; THOMAS et al., 1994b; YEUNG et
al., 1994; LAPOINTE et al., 1996). Recently, oviduct specific glycoproteins have been
shown to support bovine sperm motility (ABE et al., 1995a; SATOH et al., 1995) and
enhance oocyte penetration of hamster spermatozoa (BOATMAN and MAGNONI,
1995). Other studies revealed that binding of spermatozoa to OEC results in
extended sperm longevity (LEFEBVRE and SAMPER, 1993; ELLINGTON et al.,
1993b), improve fertilizing capacity (SMITH and YANAGIMACHI, 1991; POLLARD et
al., 1991; CHIAN and SIRARD, 1995), increased zona binding ability (ELLINGTON et
al., 1993c; ZISKIND et al., 2000) and maintained viability of spermatozoa (SMITH et
al., 1987; SMITH and YANAGIMACHI, 1990). The apical plasma membrane of
bovine OEC contains anchored proteinic factors that contribute to maintaining motility
and viability and possibly to modulating capacitation of bovine sperm (BOILARD, et
al., 2002). Exposure of sperm cells to homologous OEC or their secretory products
Review of literatures ___________________________________________________________________
10
have resulting beneficial effect on sperm survival and motility parameters during in
vitro culture (KERVANCIOGLU et al., 1994; ZHU et al., 1994; PACEY et al., 1995).
Co-culture of stallion spermatozoa with OEC monolayers maintained viability up to 4
days, while spermatozoa in medium alone didn’t survive more than 24 hours (SMITH
and YANAGIMACHI, 1990). In addition bovine sperm membrane integrity could be
maintained by co-incubating with OEC in vitro (POLLARD et al., 1991).
2.2.3 Regulation of sperm capacitation
Sperm capacitation is a necessary prelude to fertilization and constitutes a set of
changes in the plasma membrane that enable sperm to undergo the acrosome
reaction (BLEIL and WASSERMAN, 1980, 1983). This set of changes consists of
removal, addition and/or alterations of the sperm plasma membrane components.
Portion of these changes occurs in the oviduct (ADAMS and CHANG, 1962;
HUNTER and HALL, 1974; YANAGIMACHI, 1994). Studies in cattle (GUYADER and
CHUPIN, 1991), sheep (GUTIERREZ et al., 1993), hamster (SMITH and
YANAGIMACHI, 1990) and horses (ELLINGTON et al., 1993a,c) have shown that
sperm released from OEC in vitro were at least partially capacitated as determined
by induction of acrosome reaction or in vitro fertilization. Furthermore it was reported
that the in vitro sperm-oviduct interaction in swine is initiated by uncapacitated
spermatozoa binding to OEC and is continued by the induction of capacitation in
cocultured spermatozoa (FAZELI et al., 1999). Moreover, sperm capacitation was
promoted by oviductal fluid (PARRISH et al., 1989; MC NUTT and KILLIAN, 1991;
MAHMOUD and PARRISH, 1996) and bovine oestrus associated protein (KING et
al., 1994). Oviductal fluid could induce sperm capacitation by facilitating efflux of
cholesterol from the sperm plasma membrane (EHRENWALD et al., 1990). However,
the ratio of cholesterol to phospholipids was higher in fluid collected from the bovine
oviductal isthmus compared to ampullary fluid, supporting the concept that the
isthmus serves as a sperm reservoir, where cholesterol efflux would be minimized
(GRIPPO et al., 1994). OEC-conditioned medium (ANDERSON and KILLIAN, 1994;
CHIAN et al., 1995) and coculture of spermatozoa with OEC were used to promote
sperm capacitation in vitro (sheep: GUTIERREZ et al., 1993; cattle: ELLINGTON et
Review of literatures ___________________________________________________________________
11
al., 1991; GUYADER and CHUPIN, 1991; horses: ELLINGTON et al., 1993a;
humans: KERVANCIOGLU et al., 1994). Because the life span of capacitated
spermatozoa is relatively short (BEDFORD, 1983; YANAGIMACHI, 1994), regulation
of the rate of capacitation of spermatozoa stored in the oviductal isthmus could
represent an important mechanism for ensuring availability of viable spermatozoa at
the site of fertilization. It has been reported that capacitation of spermatozoa in the
female reproductive tract involves changes in the sperm plasma membrane and an
influx of calcium ions (SINGH et al., 1978; RUKNUDIN and SILVER, 1990).
DOBRINSKI et al. (1997) concluded that maintenance of low Ca2+, delay of
capacitation and prolonged viability were observed in equine spermatozoa incubated
with OEC in vitro. Changes in the sperm head membrane are also observed by SEM
during in vivo interaction with the OEC and are considered as a manifestation of the
completion of capacitation (HUNTER et al., 1991). It has been reported that equine
spermatozoa, which are released from OEC monolayers and capacitated, can bind to
the zona pellucida (ELLINGTON et al., 1993c). Moreover exposure of spermatozoa
to OEC also promoted the acrosome reaction (DE JONGE et al., 1993; EVREV et al.,
1994; GRIPPO et al., 1995). An explanation for this functional ambiguity of the
oviducts is that sperm attachment to OEC delays capacitation while OEC secretions
induce capacitation (SMITH, 1998).
2.2.4 Reduction the incidence of polyspermy
Sperm binding to OEC could limit the number of spermatozoa at the site of
fertilization, while ensuring that a sufficient number is available in the oviduct when
ovulation occurs. In hamster the ratio of sperm to eggs at the site of fertilization is
less than 1:1 until at least half the eggs are fertilized (CUMMINS and
YANAGIMACHI, 1982). When porcine sperm were surgically infused into oviducts at
the pre-ovulatory stage, a high incidence of polyspermy resulted (HUNTER, 1973;
HUNTER and NICHOL, 1988). HUNTER and LEGLISE (1971) found that polyspermy
increased after resection of the isthmic part of the oviduct in pigs. Furthermore, the
rate of polyspermy decreased when boar sperm were incubated with OEC prior to
fertilization in vitro (NAGAI and MOOR, 1990; KANO et al., 1994; DUBUC and
Review of literatures ___________________________________________________________________
12
SIRARD, 1995). Thus prevention of polyspermy may be an additional function of
sperm binding to oviductal epithelium in the reservoir.
2.3 SPERM RELEASE FROM THE OVIDUCTAL RESERVOIR
Bovine spermatozoa can remain arrested in the isthmus for up to 18 h and detached
from the epithelium near the time of ovulation and a few reaches the ampulla where
fertilization takes place (HUNTER and WILMUT, 1984; SUAREZ et al., 1990;
HUNTER, 1998). Moreover it was reported that release of spermatozoa from the
oviductal isthmus appears to be associated with ovulation (pig: HUNTER, 1981;
sheep: HUNTER et al., 1982; hamster: SMITH and YANAGIMACHI, 1989; ITO et al.,
1991). On the contrary, DE MOTT and SUAREZ (1992) suggested that release of
sperm from the isthmic reservoir is independent of ovulation. Evidence for a role of
sperm capacitation in the release of spermatozoa from the OEC has been found in
hamsters (SMITH and YANAGIMACHI, 1989; 1991), cattle (LEFEBVRE and
SUAREZ, 1996) and horses (THOMAS et al., 1995a). Moreover, DOBRINSKI et al.
(1997) reported that changes in the sperm surface characteristics associated with
capacitation trigger the release of spermatozoa from the oviductal epithelium. A third
factor implicated in the detachment of spermatozoa from OEC is the development of
hyper-activated motility in oviductal spermatozoa (KATZ and YANAGIMACHI, 1980;
SUAREZ, 1987; SUAREZ and OSMAN, 1987; GUERIN et al., 1991; DE-MOTT and
SUAREZ, 1992; SUAREZ et al., 1992). In vitro studies suggested that spermatozoa
may develop hyperactivated motility while bound to OEC (ELLINGTON et al., 1991;
1993b; POLLARD et al., 1991). DE MOTT et al. (1995) stated that fetuin interfered
with hamster sperm attachment to the oviductal epithelium by binding to the
acrosomal region of the fresh epididymal sperm. Feutin did not bind to hyper-
activated sperm. Therefore, release of sperm from the oviductal epithelium may be
associated with modification of sperm head surface that coincide with capacitation
and /or hyper-activation.
Review of literatures ___________________________________________________________________
13
2.4 IN VITRO SYSTEMS TO STUDY THE SPERM OVIDUCT
INTERACTION
2.4.1 Tissue explants
Explants of oviductal epithelium from surgically excised oviducts or from oviducts
recovered after slaughter have been used to study the interaction of spermatozoa
with OEC in pigs (RAYCHOUDHURY and SUAREZ, 1991), cattle (LEFEBVRE, 1997;
LEFEBVRE et al., 1995; 1997) and horses (LEFEBVRE and SAMPER, 1993;
THOMAS et al., 1994a). Explants cultures were also used to characterize secretion
of lipids and proteins synthesis by the OEC (HENAULT and KILLIAN, 1993;
WOLDESENBET and NEWTON, 2003) and to study ciliary’s activity (GADDUM-
ROSSE and BLANDAU, 1976). Within 30 min of disaggregation the clumps of
epithelial cells formed everted vesicles with apical surfaces facing outward,
henceforth referred to as explants (SUAREZ et al., 1991; 1998; POLLARD, 1992;
THOMAS et al., 1994a; DE MOTT et al., 1995). OEC vesicles are physiologically
more responsive model for the in vivo oviduct than are OEC monolayers (BUREAU et
al., 2002). DE PAUW et al. (2002) have developed a new in vitro method for
analysing the sperm oviductal explants binding capacity and found that bovine
sperm-oviduct interaction can preferably be investigated in oviductal explants smaller
than 20000 µm2. The later authors added that determination of the sperm oviductal
explants binding capacity could become a reliable in vitro method for predicting the
NRR of a given sire.
2.4.2 Oviductal epithelial cells monolayers (OECM)
Because of their more uniform characteristics compared to tissue explants and
because they can be stored frozen and are easier to handle, oviductal epithelial cell
monolayers (OECM) grown in culture have been widely used to study sperm-oviduct
interaction (sheep: GUTIERREZ et al., 1993; dogs: ELLINGTON et al., 1995; cattle:
ELLINGTON et al., 1991; CHIAN and SIRARD, 1995; horses: THOMAS et al., 1994b,
1995a; THOMAS and BALL, 1996; humans: BONGSO et al., 1993;
Review of literatures ___________________________________________________________________
14
KERVANCIOGLU et al., 1994; ELLINGTON et al., 1998a,b). OEC monolayers can be
maintained in serum free culture (VAN LANGENDONCKT et al., 1995) and it have
been characterized with regard to its cell morphology and protein secretion (cattle:
JOSHI, 1991; horses: ELLINGTON et al., 1993d; THOMAS et al., 1995b,c). Cilia on
OEC were generally lost after passage of OEC monolayers in culture (JOSHI, 1988,
1991; BATTUT et al., 1991; THOMAS et al., 1995c). OEC monolayers could be
passaged for up to 14 -18 passages in cattle, 10 passages in rabbits, 6 passages in
humans before reaching a crisis stage characterized by arrest of cell growth and
alterations in cell morphology. In contrast, epithelial cell monolayers from mice
couldn’t be sub-cultured (OUHIBI et al., 1989). Oviductal epithelial cell monolayers
have been shown to respond to the introduction of spermatozoa into the culture with
a change in their secretory products (ELLINGTON et al., 1993d; THOMAS et al.,
1995b). Monolayers of oviductal epithelial cells have also been used to study
formation of oviductal fluid and glucose transport in rabbits (DICKENS et al., 1993;
EDWARDS and LEESE, 1993).
2.5 SPERM CHROMATIN STABILITY
The item sperm chromatin means the sperm DNA and its adherent proteins. The
integrity of mammalian sperm DNA is of vital importance for the paternal genetic
contribution to a normal offspring and the chromatin status of the sperm is important
for successful embryo development (BEDFORD et al., 1973; EVENSON et al., 1980).
Furthermore, damaged DNA in the single sperm that fertilizes a female oocyte can
have a dramatic negative effect on the embryo development (EVENSON, 1997;
1999a,b).
2.5.1 Sperm chromatin packaging
The nuclear structures of spermatogonia, spermatocytes and early round spermatids
are similar to that observed in somatic cells. However, during mid to late
spermiogenesis the spermatid nucleus undergoes transformations by two distinct
processes. The first process involves reconfiguration of the nuclear matrix
(BENAVENTE and KROHNE, 1985; LONGO et al., 1987; BELLVÈ et al., 1990;
Review of literatures ___________________________________________________________________
15
HESS et al., 1993). The second process of nuclear reorganization involves
replacement of the somatic cell like histones firstly with transition proteins and final
addition of sperm specific protamines (CALVIN and BEDFORD, 1971; FAWCETT et
al., 1971; KUMAROO et al., 1975; MEISTRICH et al., 1976; WARRANT and KIM,
1978; BALHORN, 1982; WARD and COFFEY, 1991; GREEN et al., 1994).
Mammalian protamines are rich in arginine and cysteine residues, which form
disulfide (S-S) bonds within and among adjacent protamine molecules. Two types of
protamines, 1 and 2 have been described in sperm nuclei of eutherian mammals.
However, mature bovine sperm contain only protamine-1, which typically forms two
intramolecular, and three intermolecular S-S bonds (BALHORN et al., 1991). The
most accepted model for how protamines interact with sperm DNA predicts that
protamines lie lengthwise in the minor groove of the DNA with each positively
charged arginine residue neutralizing one negative charge of the DNA’s
phosphodiester backbone (BALHORN, 1982; WARD and COFFEY, 1991;
PIRHONEN et al., 1994) as shown in figure 1.
Review of literatures ___________________________________________________________________
16
Figure 1: Equivalent levels of DNA packaging in somatic cells (left) and sperm cells (right). In somatic cells, DNA is compacted into solenoids with about 6 nucleosomes per turn. In the sperm nucleus, protamines bind to the DNA, neutralizing its negative charge, and coiling the complex into tight circles these circles collapse into a "doughnut shaped structure." Each doughnut represents one DNA loop attached to the nuclear matrix. (WARD, 1993).
Review of literatures ___________________________________________________________________
17
Recent evidence would contradict this model and suggests that protamines bind in
the major rather than the minor groove of the DNA (HUD et al., 1993; PRIETO et al.,
1997; BREWER et al., 1999). Regardless, binding of protamines to sperm DNA
transforms the poly-anionic DNA into a stable, neutral polymer which is resistant to
chemical and physical damage and is nearly 6 times more condensed than DNA
found in mitotic chromosomes (POGANY et al., 1981). Mature mammalian
spermatozoa contain high percentages of protamines, for example human and
mouse sperm nuclei contain more than 85 % and 95 % protamines in their
nucleoprotein component respectively (DEBARLE et al., 1995). In mice, protamines
allow the mature sperm nuclei to adopt a volume 40 times less than that of normal
somatic nuclei (WARD and COFFEY, 1991). When spermatozoa migrate through the
epididymis sulphhydryl groups of the cysteine-rich protamines become oxidized
resulting in large numbers of disulphide bonds between cystine residues. Such
changes are thought to stabilize sperm nuclei (BEDFORD, et al., 1973; BEDFORD
and CALVIN, 1974). Moreover, after ejaculation zinc enters the chromatin and binds
to the free thiol groups to stabilize its quaternary structure (ARVER and ELIASSON,
1982; BJORNDAHL and KVIST, 1990). Thus stabilization of chromatin seems to
compensate for the lack of DNA-repair enzymes (MATSUDA et al., 1985). Chromatin
condensation is disturbed when lysine-rich somatic histones are not sufficiently
substituted by arginine- and cysteine-rich protamines during spermiogenesis
(MEISTRICH et al., 1978, 1976). Complete chromatin packaging is essential for
normal sperm functioning (KOSOWER et al., 1992). It has been shown that
incomplete replacement of histones by protamines is associated with male subfertility
(AUGER et al., 1990). Stabilization is not always complete, since it has been shown
that there are great differences among the spermatozoa present in any given
ejaculates (KOSOWER et al., 1992). Moreover, heterogeneity of sperm nuclear
maturity has been reported in different semen samples especially between fertile and
infertile patients (EVENSON et al., 1980).
Review of literatures ___________________________________________________________________
18
2.5.2 Factors affecting sperm-chromatin stability
The stability of sperm chromatin is not constant, as it can be clearly changed in an
individual animal within a short period of time (BALLACHEY et al., 1987; GOGOL et
al., 2002).
2.5.2.1 Age of the semen donors and aging of spermatozoa
Age of semen donors appears to be related to a significant increase in sperm DNA
fragmentation (SPANO et al., 1998; EVENSON et al., 2002). Comparison of
chromatin structure of sperm from two groups of bulls aged 14 months and 4 years
indicates that this parameter improves with the bull's age (KARABINUS et al., 1990).
In addition, studies involving a large group of men showed the age of semen donors
to be strongly correlated to sperm chromatin structure (SPANO et al., 1998).
Moreover, decreased sperm chromatin stability was found in ejaculates taken from
male rabbits less than 5 months and more than 20 months of age (GOGOL et al.,
2002).
A further factor which affects the sperm chromatin stability is the long sexual
abstinent (EVENSON et al., 1991; SPANO et al., 1998). The semen samples of
rabbits, bulls and sheep, which were collected outside of the breeding season, had
showed increased chromatin instability and less fertility than those collected during
the breeding season. This might be due to over-maturation of spermatozoa during
long storage in the epididymis (MILLER and BLACKSHAW, 1968; SALISBURY and
HART, 1970; RODRIGUEZ et al., 1985). Furthermore, sperm aging in vitro also
results in increased susceptibility of sperm DNA to denaturation. ESTOP et al. (1993)
demonstrated that mouse sperm aged in vitro showed chromatin denaturation within
one hour of incubation at room temperature.
2.5.2.2 Temperature
A further essential cause for the increased occurrence of unstable sperm chromatin
is an increase of both internal body temperature and ambient temperature
(THIBAULT et al., 1966; STONE 1977). The patients suffering from cryptorchidism
(the testicles lie in the abdominal cavity) are infertile, because the higher abdominal
Review of literatures ___________________________________________________________________
19
temperature disturbs the spermatogenesis process (CREW, 1922). Moreover, the
portion of spermatozoa with unstable chromatin was clearly increased in individuals
with feverish illnesses and showed likewise fertility disturbances (GUNN et al., 1942;
EVENSON et al., 2000).
2.5.2.3 Cryoconservation
The influence of cryoconservation procedure on the sperm chromatin status is
controversially discussed. EVENSON et al., (1994) estimated that neither the process
of the cryoconservation nor the shock freezing of ejaculates had an influence on the
ultrastructure or stability of sperm chromatin, whereas others observed a degradation
of the chromatin stability particularly with subfertile individuals (HAMMADEH et al.,
1999; 2001; BLOTTNER et al., 2001). KARABINUS et al. (1990) stated that
incubation of bull sperm in cryoprotectant media increased the susceptibility of DNA
to denaturation in situ within 30 minutes. ROYERE et al. (1988) and HAMAMAH et al.
(1990) claimed that a relationship existed between an "over-condensation" state for
frozen-thawed sperm chromatin and a lower conception rate for human semen after
cryostorage. ROYERE et al. (1991a) suggested that freeze-thawing procedures
might alter the DNA / nuclear protein relationships and impair the fertilizing ability of
human sperm. In addition, frozen-thawed boar spermatozoa showed significantly
increased (P < 0.05) chromatin compactness compared to fresh spermatozoa.
Moreover CORDOVA et al. (2002) found that the percentage of spermatozoa with
unstable chromatin was significantly (P < 0.05) higher in frozen semen samples than
that found in fresh semen.
2.5.2.4 Reactive oxygen species (ROS)
Reactive oxygen species (ROS) are harmful to sperm at elevated levels (JONES and
MANN, 1973; ALVAREZ et al., 1987; AITKEN et al., 1989a,b; 1992; D'AGATA et al.,
1990; AITKEN and Fisher, 1994; CUMMINS et al., 1994; BECKMAN and AMES,
1997; ARMSTRONG et al., 1999; EVENSON et al., 2002) and are a major cause of
damage to sperm DNA (GAGNON et al., 1991). The major sources of ROS in diluted
semen incubated at ambient temperature are oxidative de-amination of aromatic
Review of literatures ___________________________________________________________________
20
amino acids by aromatic L-amino acid oxidase released from dead and damaged
sperm (SHANNON and CURSON 1972; 1981), mitochondrial respiration (AITKEN
and CLARKSON, 1987), and seminal leukocytes (AITKEN et al., 1992;
KESSOPOULOU et al., 1992; ALVAREZ et al., 2002). Because sperm are almost
devoid of cytoplasm, they possess only very low amounts of the ROS-scavenging
enzymes that protect somatic cells from oxidative damage. Moreover DNA repair
enzymes are apparently inactive in mature sperm making these cells more
susceptible to oxidative attack (HUGHES et al., 1998). Functional sperm rely on the
tight packing of their DNA around protamines, which reduces exposure to free
radicals and on antioxidants present in the seminal plasma for protection from
oxidative damage (HUGHES et al., 1998). During in vitro manipulation of sperm
samples oxidative damage to sperm DNA can be alleviated by supplementing the
diluent with antioxidants (HUGHES et al., 1998), ROS-degrading enzymes and
elimination of oxygen from the diluent (SHANNON and CURSON, 1982).
2.5.2.5 Trace elements and other factors
Zinc and copper are trace elements, which play an important role in the stability of
sperm cells chromatin by stabilization of the free thiol group. The Prostate gland
secretion is rich with zinc, so that the sperm chromatin is protected when mixed with
seminal plasma during ejaculation. A lack of zinc leads to increased susceptibility of
the sperm chromatins to in situ denaturation (BLAZAK and OVERSTREET, 1982;
RODRIGUEZ et al., 1985). Some therapeutically used chemicals (SHALET, 1980;
EVENSON et al., 1999), environmental pollution stress (WYROBEK et al., 1997;
LEMASTERS et al., 1999; PERREAULT et al., 2000; SELEVAN et al., 2000),
cigarette smoking (SPANO et al., 1998) and cancer diseases (EVENSON and
MELAMED, 1983; EVENSON et al., 1984; FOSSA et al., 1997) are also factors,
which negatively affect the stability of sperm chromatin. It is noteworthy that a partial
decondensation state of human sperm chromatin may occur during in vitro
capacitation. However, beyond some level of decondensation the fertilizing ability
could be altered (ROYERE et al., 1991b).
Review of literatures ___________________________________________________________________
21
2.5.3 Evaluation of sperm chromatin stability
In assessing semen quality, animal and human fertility clinics typically measure
sperm density, motility and morphology. Clinics rarely measure sperm DNA integrity,
primarily because they are unaware of the availability a rapid, reliable and practical
test. The methods of studying sperm chromatin status includes: aniline blue (AB),
which indicates the presence of excessive histones (TERQUEM and DADOUNE,
1983), Chromomycin A3 (CMA3), which shows protamine deficiency (IRANPOUR et
al., 2000), comet assay, which shows extent of DNA fragmentation (HUGHES et al.,
1999) and acridine orange (AO), which reflects chromatin resistance to denaturation
(TEJADA et al., 1984).
2.5.3.1 Sperm chromatin structure assay (SCSA)
Acridin orange (AO) intercalates into double-stranded (ds) DNA as a green
fluorescing monomer and binds to single-stranded (ss) DNA as a red fluorescing
aggregate when excited by a blue laser light (488nm) (ICHIMURA et al., 1971). The
SCSA was developed to measure sperm DNA susceptibility to in situ acid induced
denaturation by quantifying the metachromatic shift from green fluorescence of AO
bound to ds-DNA to red fluorescence emitted by AO bound to ss-DNA (EVENSON et
al., 1980). Two DNA denaturation methods using AO were used, one combined from
the earlier recommendations of ROSCHLAU, (1965) and RIGLER, (1966) (RRAO
method), which used earlier for in situ detection of apoptotic cells, and the other
method suggested by TEJADA et al. 1984 (TAO method) which used for sperm cells.
The SCSA is an adaptation of the two-steps AO procedure originally designed by
DARZYNKIEWICZ and colleagues (1975) for simultaneous measurements of DNA
and RNA content in somatic cells. Whatever minute amounts of RNA may be present
in a mature sperm do not interfere with SCSA data. It is of interest, but not
understood that this procedure denatures protamine associated DNA in sperm but
does not denature somatic cell DNA associated with histones (EVENSON et al.,
1985).
Review of literatures ___________________________________________________________________
22
2.5.3.2 Modified fluorescence microscopical SCSA (mf-SCSA)
TEJADA and co-workers (1984) developed the first modification of the SCSA for the
conventional fluorescence microscope to eliminate the necessity of flowcytometry for
the assessment of human sperm chromatin stability with AO fluorescence. This
simplified method termed acridin orange TEJADA (AOT) and based upon the same
principles as SCSA, but relies on human visual interpretation of the fluorescent
characteristics of AO intercalated into the sperm nucleus. The major benefit of
microscopical approach to SCSA is a simultaneous evaluation of sperm chromatin
status and morphology. ANGELOPOULOS et al. (1998) observed a high consistency
between the mean percent of morphologically normal spermatozoa and the percent
of green sperm determined through AOT. Moreover, DOBRINSKI et al. (1994) found
a significant correlation between red fluorescing cells and the percentage of pyriform
heads and vacuoles. However, they also found that high numbers of abnormally
condensed nuclei could be detected in the absence of other defects. Several
laboratories have used the AOT technique to assess male fertility potential (IBRAHIM
and PEDERSEN, 1988; ROUX and DADOUNE, 1989; CLAASSENS et al., 1992).
Significant correlations were recorded between red fluorescence and abnormal
sperm morphology, but not between red fluorescence and motility (TEJADA et al.,
1984; IBRAHIM and PEDERSEN, 1988). This suggests a potential infertility factor in
spermatozoa that is removed from sperm viability alone. There have been problems
reported with the interpretation of AO fluorescence on sperm cells using AOT.
Rapidly fading fluorescence and indistinct colour are most commonly associated with
this technique (DURAN et al., 1998; CLAASSENS et al., 1992). Others have modified
the technique to enhance the stability of the stain across a variety of species
(DOBRINSKI et al., 1994; BELETTI and MELLO, 1996). KOSOWER et al. (1992)
noticed that the acridine orange fluorescence of sperm nuclei is determined by the
thioldisulfide status of DNA-associated protamines. Their results indicate that sperm
nuclei treated with acetic alcohol show a green fluorescence when their nuclear
protamines are rich in disulfide bonds and show red fluorescence when their
protamines are poor in disulfide bonds (KOSOWER et al., 1992). This suggests that
the structure of chromatin is important for controlling the interaction of AO with DNA.
Review of literatures ___________________________________________________________________
23
This finding strengthens the SCSA argument that varying degrees of susceptibility to
acid denaturing conditions are indicative of abnormal chromatin structure (EVENSON
et al., 1985). Perhaps the use of thiol-protectant chemicals such as dithiothreitol or
2-mercaptoethanol may help stabilize the chromatin after acid treatment and
minimize the present constraints associated with the AOT technique, i.e. rapid
quenching and/or shifts in fluorescence color over time.
2.5.4 The sperm chromatin status and fertility
The SCSA has been used to establish a relationship between fertility and sperm
chromatin stability in cattle (EVENSON et al., 1980; BALLACHEY et al., 1988;
DOBRINSKI et al., 1994), humans (EVENSON et al., 1980; EVENSON, 1999a),
swine, (EVENSON and JOST, 1994), mice, (BALLACHEY et al., 1986) and horses
(KENNEY et al., 1995). Several studies have shown strong negative correlations
between the SCSA variables and bull fertility as measured either by non-return rate
of the female (KARABINUS et al., 1990) or the competitive index based on
heterospermic performance among bulls (BALLACHEY et al., 1988).
Chromatin defected spermatozoa are able to fertilize the oocyte, however results in
high rate of early embryonic mortality (TEJADA et al., 1984; GORCZYCA et al., 1993;
DARZYNKIEWICZ et al., 1997; EVENSON et al., 2000). EVENSON et al. (1980)
found that sperm of bulls, mice and humans with low or questionable fertilization
ability shows a significant reduction of the DNA resistance to in situ denaturation
compared with semen from normal fertile individuals. KENNEY et al. (1995) stated
that morphologically abnormal spermatozoa of subfertile horses possess nearly
unstable chromatin. Furthermore it was estimated that many of the morphologically
normal sperm of subfertile individuals exhibited increased susceptibility of their DNA
to denaturation in situ indicating that the entire sperm population of these ejaculate is
qualitatively inferior (TEJADA et al., 1984; BALLACHEY et al., 1988; LOPES et al.,
1998). It was reported that reduced sperm chromatin stability as measured by SCSA
correlates strongly with DNA strand breaks (ARAVINDAN et al., 1997) and sub-
fertility in bull, human and boar (BALLACHEY et al., 1987; 1988; EVENSON et al.,
1994; 1999). Moreover it was estimated that DNA fragmentation of human
Review of literatures ___________________________________________________________________
24
spermatozoa is negatively correlated with in vitro fertilization outcome (SUN et al.,
1997; LOPES et al., 1998; EVENSON et al., 1999). It was concluded that sperm
morphology and protamine deficiency independently affect fertilization rate
(ESTERHUIZEN et al., 2000a,b; NASR-ESFAHANI et al., 2001). On the contrary,
SPANO et al. (1999) observed no relationship between chromatin stability and
fertilization rate, suggesting that this difference was due to methodology of acridin
orange assessment. ANGELOPOULOS et al. (1998) stated that AO staining did not
predict fertilization efficiency or pregnancy outcome in IVF cycles.
2.5.5 Efficiency of SCSA to predict fertility
SCSA data on thousands of semen samples from bulls (BALLACHEY et al., 1987;
1988; EVENSON, 1999a,b), stallions (LOVE and KENNEY, 1998; EVENSON and
JOST, 2000), human (SUN et al., 1997; LOPES et al., 1998) and boars (EVENSON
et al., 1994) showed the clinical value of this assay for animal fertility assessment.
Some investigators suggested that sperm from subfertile men showed an increase in
red fluorescence (EVENSON et al., 1980; TEJADA et al., 1984; LIU and BAKER,
1992). Moreover, it was concluded that spermatozoa that fertilize the oocytes in vivo
and in IVF were limited to whose nuclei exhibited green AO fluorescence (HOSHI et
al., 1996). The SCSA was found to be a useful indicator of fertility because of its
ability to evaluate a parameter of the spermatozoa that cannot be assessed by
traditional tests of sperm quality such as motility and morphology (LOVE and
KENNEY, 1998). In addition, the SCSA may be a valuable complement for routinely
practiced microscopic evaluation of sperm morphology of AI bull semen
(JANUSKAUSKAS et al., 2000), since the morphologically abnormal spermatozoa
might possess DNA strand breaks (SAKKAS et al., 1999), as well as abnormal
chromatin structure (SAILER et al., 1996). In other studies a weakly correlation
between SCSA and conventional spermatological parameters were recorded
(GORCZYCA et al., 1993; DARZYNKIEWICZ et al., 1997; BOCHENEK et al., 2001;
EVENSON et al. 1991, 1999). Semen samples with normal conventional parameters
may have very poor DNA quality that contributes to infertility. Therefore, the SCSA
test offers additional clinical information not provided by conventional semen analysis
Review of literatures ___________________________________________________________________
25
alone. On the contrast, AO staining did not predict fertilization efficiency or pregnancy
outcome in IVF cycles (ANGELOPOULOS et al., 1998). Furthermore, the ability of
the SCSA to predict fertilization and pregnancy outcome after in vitro fertilization
(IVF) is controversially discussed (CLAASSENS et al., 1992; LIU and BAKER, 1992;
EGGERT-KRUSE et al., 1996a; HOSHI et al., 1996).
2.6 SPERM MEMBRANE INTEGRITY
The sperm plasma membrane is the outer cell structure that acts as a physiological
barrier and its functional and structural integrity are required for their normal
activities.
2.6.1 Structural integrity of sperm membrane
Several methods have been used to distinguish between viable and non-viable cells.
The sperm vital stains such as eosin/nigrosin (EN) and trypan-blue (TB) have been
used for decades to evaluate plasmalemma (sperm outer membrane) integrity
(HANCOCK, 1951; MAYER et al., 1951; SWANSON and BEARDEN, 1951;
HACKETT and MACPHERSON, 1965). Staining with EN has been combined with
Giemsa and this method is reliable and simple enough for routine work (TAMULI and
WATSON, 1994). In addition, nucleic acid stains such as Hoechst 33258 have been
combined with fluoresceinated lectins (MORTIMER et al., 1990; CASEY et al., 1993;
VALCARCEL et al., 1997), or with other fluorescent probes such as chlortetracycline
to assess capacitation and acrosomal exocytosis (FRASER et al., 1995; WANG et
al., 1995). Combination of fluorescent probes can be used to assess sperm viability
(GARNER et al., 1986). Alternatively by combining a probe that distinguishes
between viable and non-viable cells with another probe that stains the acrosomal
contents or outer acrosomal membrane, both sperm viability as well as acrosomal
status can be assessed (TAO et al., 1993; MAXWELL and JOHNSON, 1997;
COOPER and YEUNG, 1998; HARKEMA et al., 1998).
The fluorescent stain Propidium Iodide (PI) is a DNA-specific red fluorescent stain
that does not penetrate intact plasmalemma and can be combined with fluorescent
stains that penetrate also intact plasma membrane, like carboxyfluorescein diacetate
Review of literatures ___________________________________________________________________
26
(CFDA; GARNER et al., 1986; HARRISON and VICKERS, 1990) and SYBR-14
(GARNER and JOHNSON, 1995). GARNER et al., (1994) developed a double,
supravital stain consisting out of PI and SYBR14. The two stains, SYBR-14 (a green,
membrane-permeable stain) and PI (a red, membrane-impermeable counter stain)
have the same cellular target (sperm DNA). A direct comparison among vital stains
and the HOST for their ability to predict fertility has not been reported, albeit results of
the HOST and SYBR/PI staining have been shown to be well correlated with non
return rates (NRR) after artificial insemination (AI) in two independent studies
(CORREA et al., 1997; JANUSKAUSKAS et al., 2000).
2.6.2 Functional integrity of sperm membrane
The ability of the sperm tail to swell and/or coil in the presence of the hypoosmotic
solution demonstrates that the influx of water across the membrane occurs normally
(JEYENDRAN et al., 1984; KIEFER et al., 1996; BILJAN et al., 1996). DREVIUS and
ERIKSSON (1966) and DREVIUS (1972) demonstrated that sperm cells do swell in
hypo-osmotic environment and that over a relatively wide range of tonicities they
behave as perfect osmometers (swelling is proportional to the degree of
hypotonicity). This phenomenon indicates the normal plasma membrane integrity of
spermatozoa. Furthermore, the percentage of swollen cells within a sperm
subpopulation has been suggested to be an indicative parameter for the membrane
biochemical activity and fertility of human spermatozoa (JEYENDRAN et al., 1984).
2.6.2.1 The hypo-osmotic swelling test (HOST)
In the classic hypo-osmotic swelling test, the cells are classified subjectively by
morphologic evaluation as swollen or non-swollen using phase contrast microscopy
after fixation and cells with swollen tails are supposed to be membrane-intact
(JEYENDRAN et al., 1984). The HOST provides information regarding the membrane
integrity of the sperm tail, but it has been shown that the membranes at the head and
the tail compartments have different liability (HAMMERSTEDT et al., 1979). Since
spermatozoa appear to behave as perfect osmometers (DU et al., 1994; GILMORE
et al., 1996), changes in cell osmole content, as well as the distribution of the
Review of literatures ___________________________________________________________________
27
osmotically active and inactive cells within a sperm population might be revealed as
volume changes of sperm cells under iso-osmotic conditions. However, information
about changes in cell osmole content might be more detectable under hypo-osmotic
stress, since this would induce a rapid increase of cell volume due to the high water
permeability of the spermatozoa (GILMORE et al., 1996).
2.6.2.2 Modified hypo-osmotic swelling test (m-HOST)
An alternative version of HOST is the m-HOST in which not only the number of
swollen sperm but also both the extent of swelling and volume distributions of the
sperm cell populations can be objectively measured by means of an electronic cell
counter called CASY1 (PETZOLDT, 1988; ENGEL and PETZOLDT, 1994;
PETZOLDT and ENGEL, 1994). CASY1-measurements are based on the pulse
frequency analysis providing high precision and resolution of data. It is possible to
differentiate between sperm cell subpopulations in the cell volume distribution curves.
Among volumetric parameters, which can be derived from CASY1, the relative
volume shift (RVS) of the modal value of the volume distribution curve and regulative
volume decrease (RVD). RVS was suggested to be an indicative parameter of the
membrane integrity of human sperm (ENGEL and PETZOLDT 1994; PETZOLDT and
ENGEL 1994). Intact cells swell in response to hypo-osmotic conditions, following
that a cell volume reduction occurs (STRANGE et al., 1996). RVD under hypo-
osmotic conditions as described for somatic cells has been documented in bovine
sperm (KULKARNI et al., 1997). The RVD is an important physiological function as
shown by studies of sperm from c-ros tyrosine kinase receptor knockout mice
(YEUNG et al., 1999).
2.6.3 Efficiency of the HOST to predict fertility
Different clinical studies indicate that the HOST as a single assay is not sufficient to
predict the fertilizing capacity of an ejaculate (JEYENDRAN et al., 1992; VAN DEN
SAFFELE et al., 1992; SALLAM et al., 2003). Furthermore, no significant correlation
was observed between sperm swelling and in vitro sperm fertilizing capacity as
assessed by the zona-free hamster oocyte penetration assay (SMITH et al., 1992).
Review of literatures ___________________________________________________________________
28
Studies have been performed to evaluate the correlation of subnormal HOST scores
with other semen parameters that are believed to be predictive of fertilization
potential, albeit with various conclusions (CHAN et al., 1985; 1991; WANG et al.,
1988; COETZEE et al., 1989; MORDEL et al., 1989; FUSE et al., 1991; OSTERHUIS
et al., 1996; BUCKETT, 2003). Combining HOST results with other spermatological
parameters (e.g. motility and vitality) was useful in order to improve correlations with
the fertility rate in vitro (VAN DER VEN et al., 1986; MCCLURE and TOM, 1991;
RAMIREZ et al., 1992) and conception rate in human (ASCHKENAZI et al., 1992).
Although a couple of studies have found lower fertilization rates with subnormal
HOST scores (VAN DER VEN et al., 1986; AVERY et al., 1990; HAUSER et al.,
1992; ABU-MUSA et al., 1993; TARTAGNI et al., 2002), the majority have found
HOST to be one of the least useful tests to determine low fertilization potential of
sperm (BARRATT et al., 1989; SJOBLUM and COCCIA, 1989; AVERY et al., 1990;
CHAN et al., 1990; ENGINSU et al., 1992; KIEFER et al., 1996). Recently a defect in
sperm volume regulation has been identified as the cause of sterility in the protein
tyrosine kinase receptor c-ros knockout mouse (YEUNG et al., 1999, 2000). Positive
correlations between the results of HOST and hamster egg penetration assay were
observed for specimens with high or low fertility rates (i.e. ejaculate with low swelling
rate demonstrated low fertility), but not always in the middle range of rates (OKADA
et al., 1990). Moreover, the HOST can assist in evaluating semen quality, judged by
the fertilization rate in an in vitro fertilization program (YAVETZ et al., 1995). It was
reported that the HOST was not correlated with percentage of pregnant mares but
showed a tendency to correlate with the number of services per pregnancy; therefore
it could be an additional method for evaluating stallion fertility (NEILD et al., 1999;
2000). MCCLURE and TOM (1991) found no significant correlations between HOST
and fertility for spermatozoa from infertile men. Moreover, in cattle, a preliminary
report using five bulls found that the hypo-osmotic swelling test was not related to IVF
success (ROTA et al., 2000). It is noteworthy that the HOST can accurately evaluate
viability in fresh human spermatozoa but not in cryopreserved ones (ESTEVES et al.,
1996).
Materials and Methods ___________________________________________________________________
29
3 MATERIALS AND METHODS
The used chemicals and reagents from which the buffers, solutions, and media were
made as well as all labour objects are presented in an appendix.
This study was applied at Institute for Reproductive Medicine, School of Veterinary
Medicine HANNOVER, Bünteweg 15, D-30559 HANNOVER, GERMANY and
Institute of Animal Science and Animal Husbandry, Federal Agricultural Research
Centre (FAL), 31535 NEUSTADT-MARIENSEE, GERMANY in the period from
September 2000 to April 2004.
3.1 SEMEN SOURCE
The Rinderproduktion Niedersachsen (NORDRIND), GmbH BREMEN - HANNOVER,
Lindhooper Str.110, 27283, VERDEN, generously provided the straws of
cryopreserved semen from 30 bulls. The spermatozoa were processed and packed in
0.25 ml capacity mini-straws (approximately 20-30 x 106 sperm/ straw) and stored at
-196 oC in liquid nitrogen.
3.2 HANDLING OF FROZEN-THAWED SEMEN
Unless otherwise stated elsewhere, individual straws were thawed rapidly in a warm
water bath at 38 oC for 30 sec. Straws were dried thoroughly to avoid the possibility
of water contacting the semen when the straw is opened. Moreover fluctuations in the
temperature of thawed semen were avoided to minimize the risk of chilling injury.
3.3 TRADITIONAL SPERMATOLOGICAL PARAMETERS
3.3.1 Motility parameters
3.3.1.1 Subjective sperm motility
Individual straws from 90 ejaculates (three ejaculates per bull) were thawed and the
contents of each straw were separately transferred into a pre-warmed 1.5-ml
Materials and Methods ___________________________________________________________________
30
eppendorf plastic tube. About 5-µl aliquot of thawed semen was placed on a pre-
warmed glass slide then covered with a pre-warmed coverslip (18x18 mm).
The prepared slides were viewed under 160x magnifications using a phase-contrast
microscope equipped with a 38 oC warmed stage (Zeiss, JENA / GERMANY).
Progressive forward- and local- as well as non-motile spermatozoa were estimated to
the nearest 5 %. The rest of the semen samples were used for sperm morphology
assessment.
3.3.1.2 Assessment of motility using cell motion analyzer (CMA)
Computer-assisted sperm motility analysis was performed using Stromberg Mika Cell
Motion analyser (SM-CMA, Strömberg-Mika; Bad FEILNBACH, GERMANY) as
described previously by RODRIGUEZ-MARTINEZ and BERROSTEGUIETA (1994).
For motility analysis, about 8 µL aliquots of thawed semen were placed on a pre-
warmed (38 oC) Mika cell counting chamber (10 µm depth) and covered with a
special cover-slip, then the spermatozoa were visualized under a phase contrast
objective (Ph 2) at 200x magnification. For each attempt, several microscopic fields
(sequences) were analysed including at least 200 spermatozoa. The proportion of
total motile (TM) and linearly motile (LM) spermatozoa, straight-line velocity (VSL,
µm/s), curvilinear velocity (VCL, µm/s), and the average-path velocity (VAP, µm/s)
were determined. The computer settings were adjusted according to the
manufacturer’s instructions as follows: number of frames per analysis: 32, time
between two video half pictures for detection of immotile objects: 20 ms, cell size
range: 35-300 pixels, threshold value for velocity to be classified as immobile objects:
10 µm/sec, threshold value for velocity to be accepted as locally motile spermatozoa:
25 µm/sec, maximum value for linearity, 90 %, minimum number of frames, 15,
velocity class width, 5 µm/sec, maximum radius for circles: 25 µm.
3.3.2 Morphological abnormalities of spermatozoa
In this experiment, 90 ejaculates from 30 bulls (3 ejaculates per bull) were examined.
The frequencies of proximal and distal protoplasmic droplets, loose heads, acrosomal
Materials and Methods ___________________________________________________________________
31
abnormalities, abnormal mid-pieces and coiled tails were estimated after counting
200 spermatozoa in wet smears.
About 160 µl aliquots of thawed semen were transferred into Eppendorf tubes
containing 250 µl formol-citrate 4 %. After good mixing, wet smears were made by
placing about 2 µl of semen mixture on a glass slide then gently covered with a cover
slip. The prepared wet semen smears were examined using phase contrast (Ph 2)
microscopy at 1000x magnification under oil immersion lens. Sperm cells
abnormalities were classified according to KRAUSE (1965) as shown in figure 2.
Materials and Methods ___________________________________________________________________
32
Acrosome abnormalities 13) Retro-axial tail attachment
1) Swelled acrosome 14) Proximal protoplasmic droplet
2) Acrosome in detaching Mid-piece abnormalities
3) Detached acrosome 15) Protoplasmic droplet
4) Deformed acrosome Main-&end-tail piece abnormalities
5) Persistent acrosome granulome 16) Looped tail
Head abnormalities 17) Rolled tail
6) Deformed 18) Tail rolled around the head
7) Small 19) Distal protoplasmic droplet
8) Narrow 20) Rudimentary Tail
9) Dwarf Doubled Abnormalities
10) Large 21) Twin head and one tail
Neck abnormalities 22) One head and Twin tail
11) Broken neck (detached head)
12) Paraxial tail attachment
Figure 2: Forms of morphologically abnormal bull spermatozoa (KRAUSE 1965).
0) 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11)
12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22)
Materials and Methods ___________________________________________________________________
33
3.4 ADVANCED SPERMATOLOGICAL PARAMETERS
3.4.1 Viability assessment using LIVE/DEAD® Sperm Viability Kit
Both morphological integrity of sperm membrane (sperm viability) and mitochondrial
activity of thawed spermatozoa were evaluated using LIVE/DEAD® Sperm Viability
Kit (Molecular Probes, Mol. Biol. Tech., GÖTTINGEN). It is composed of SYBR-14,
which is a newly developed fluorescent nucleic acid stain, maximally absorbs at 488
nm and emits at 518 nm when bound to DNA of alive spermatozoa. It stains the
nuclei of living sperm bright green and Propidium Iodide stain (PI) which stains the
nucleic acid molecules of dead and membrane damaged spermatozoa red by
intercalating into them. PI excites at 536 nm and fluoresces at 617 nm. The method
described by GARNER and JOHNSON (1995) with some modifications was used as
a model in this experiment.
Hepes buffer saline (HBS, see appendix) was prepared, then divided into 10 ml
portions in closed plastic test tubes and kept deep frozen (-23 oC) till used. At the day
of the experiment, one portion of the deep frozen HBS was thawed at room
temperature. To each 10 ml portion, 0.0238 g Hepes and 0.01 g Bovine Serum
Albumin (BSA) were being added, after that the pH was adjusted to 7.4 with 0.1 N
NaOH. 500 µl aliquots of the final HBS solution were transferred to pre warmed
Eppendorf tubes. The SYBR-14 / PI working solution was thawed at room temp.
Individual frozen semen straws from each ejaculate were thawed in warm water bath
(38 oC / 10 sec) and the contents were transferred to the pre-warmed 500-µl HBS
aliquots. From the HBS-semen mixture, 500 µl aliquots were taken into another pre-
warmed Eppendorf and mixed with 0.17 µl SYBR-14 and 2 µl PI (10 µM SYBR-14
and 120 µM PI). The samples were then gently mixed and incubated at 38 oC for 15
min. The semen smears were made in dark room by placing 20 µl aliquot of stained
semen on a warm slide and smeared with the aid of another warm slide. The smears
were quickly air dried and evaluated as soon as possible using a phase contrast
florescence microscope (Phase 2), at 200x magnifications and 490 nm excitation
filter (Leitz Laborlux-11; JENA, GERMANY). With the aid of digital camera (Olympus,
Materials and Methods ___________________________________________________________________
34
HAMBURG) and Software analysis® 3.0 (Soft Imaging System GmbH, MÜNSTER),
several fields were photographed and saved for later evaluation. A minimum of 200
spermatozoa were counted and classified as alive and membrane intact when
stained green or as dead and membrane damaged when stained green–red or just
red.
3.4.2 Assessment of the functional sperm membrane integrity using
modified hypo-osmotic swelling test (m-HOST)
Biochemically active sperm is required for the process of capacitation, acrosome
reaction and binding of the sperm cell to both oviduct epithelium and oocyte surface.
The frequency of frozen thawed bull spermatozoa with functionally intact plasma
membranes can be determined using simple and practical osmotic resistance tests
based up on their behaviours when exposed to hypo-osmotic solutions
(JEYENDRAN et al., 1984). An alternative version of this test is the modified hypo-
osmotic swelling test (PETZOLDT, 1988; PETRUNKINA et al., 2001c,d).
Electronic cell counter and analyser system ´´CASY1´´ (Coulter technique employing
the “pulse area analysis” signal-processing technique; Schärfe System
REUTLINGEN, GERMANY) was used for assessment of the functional integrity of
sperm membrane (figure 3).
Materials and Methods ___________________________________________________________________
35
Figure 3: CASY1 (Cell counter and Analyzer System), Model TTC.
3.4.2.1 General physical principles of measurement
The suspended spermatozoa are introduced into the measuring unit through a
capillary of predefined geometry at a constant-stream velocity. During the
measurement, an electric current is supplied to the capillary via two platinum
electrodes, so the capillary filled electrolyte has a defined electrical resistance. While
passing through the capillary, the cells displace electrolyte solution in proportion to
cell volume. Because of the intact cells have isolation properties, resistance along the
capillary rises and producing signal. This signal is scanned by CASY1 with a
frequency of one million measurements per second in a low voltage field. CASY1
captures the amplitude and width of the pulse and determines the integral of the
measuring signal (pulse area analysis). The resulting signal of every individual cell is
analyzed in area, height, width and course of time. The cell signals are cumulated
and assigned in a calibrated multi-channel analyzer that has 512,000 channels.
Computer
Keyboard
Waste bottle
Supply Bottle
Disk Drive
Precision measuring Capillary External Electrode
Measurement Module
Pressure System
LED Indicator lamp
Base Plate
Monitor
Main Current Supply
Sample Socket
Upstream Tube
Mouse
Materials and Methods ___________________________________________________________________
36
According to Ohm’s law:
The electric voltage (U) = electric resistance(R) x electric current (I).
Because of the stability of the electric current (I), any changes in the electric
resistance will be accompanied by changes in the electric voltage. U= f x R. While
the electrolyte has a defined electrical resistance and the cells displace electrolyte
solution in proportion to cell volume, so that the volume of the cells was derived from
the electric resistance (R) = f x V.
From it, this equation resulted: U=f x V.
3.4.2.2 Calibration of CASY1 by latex beads
Because the two test solutions (iso-osmotic and hypo-osmotic) had different electrical
conductivities due to different concentrations of electrolyte, it was necessary to apply
a correction factor for the data recorded under hypo-osmotic conditions. This factor
was obtained by comparison of volume measurements of standard latex beads (3.4
µm in diameter, Sigma AG) in three HBS solutions of different osmolalities (150-,
300-, 450 mOsmol/Kg-1) at 38 oC. The volumetric parameters of latex beads
measured at different osmolalities were fitted to the linear regression model and the
obtained factor was used to calculate the real cell volume under hypo-osmotic
conditions. The calculated correction factors were 1.16 for modal values under hypo-
osmotic conditions (180 mOsmol/kg-1) and 1.11 for mean sperm cell volume.
3.4.2.3 Preparation of semen samples
Individual frozen semen straws from each ejaculate were thawed in water bath. The
thawed semen was passed through discontinuous Percoll gradients.
Preparation of discontinuous Percoll® gradients:
Percoll® (Pharmacia, UPPSALA, SWEDEN) gradients were prepared according to
the protocol described by HARRISON et al., (1993), (see appendix). The washing
method was carried out according to the protocol of PARRISH et al., (1995) with
some modification. Briefly, in obliquely positioned (45o
angle), 15 ml capacity, conical
bottom glass test tube, 2 ml 90 % Percoll was placed, and over these, another 2 ml
45 % Percoll was smoothly flowed on the inner side of the tube, then allowed
Materials and Methods ___________________________________________________________________
37
warming up. The content of each straw was carefully placed on the top of the
prepared discontinuous Percoll layers, and then centrifuged for 15 min at 700 g
(1200 rpm). After that Percoll layers were carefully withdrawn and the sperm pellets
were resuspended, each in 0.3 - 0,5 ml warm (39oC) sperm-TALP (Tyrode Albumin
Lactate Pyruvate medium; PARRISH et al., 1988) presented in Eppendorf tubes, and
then embedded in cavities found in a thick sponge at room temp (figure 4).
Figure 4: Washing of frozen-thawed bull semen through a discontinuous percoll gradient, a) before centrifugation; b) after Centrifugation.
From the preparatory work and other publications (PETRUNKINA et al., 2001c,d;
PETRUNKINA and TÖPFER-PETERSEN, 2000) it has been found that the best
sperm volume distribution curves were obtained when the sperm cell concentration
per 200 µl measuring sample volume was between 20,000 and 40,000 sperm cell.
Therefore, the sperm cell concentration was determined using Thoma cell counting
chamber to calculate how many µl semen suspensions will be needed for 6 ml HBS
to obtain a final concentration of 100,000-200,000 sperm / ml (i.e. 20,000-40,000
sperm /200 µl sample measuring volume).
a b
Materials and Methods ___________________________________________________________________
38
3.4.2.4 Volumetric measurement
Hepes buffered saline (HBS) without polyvinyl alcohol and polyvinyl pyrrolidon was
used as sperm incubation medium in the current experiment (see appendix).
Two osmolalities were used, iso-osmotic (300 mOsm kg-1) and hypo-osmotic (180
mOsm kg-1). The pH of both solutions was adjusted to 7.4 using 1N NaOH solution,
and then passed through a 0.22-µm � sterile filter (Sartorius, AG, GÖTTINGEN,
GERMANY) before use.
At the day of experiment, 10 ml capacity snap-cap glass vials (Schärfglas: Landgraf
Laborgeräte, Langenhagen) were labeled with the name of bull, osmolarity, duration
of incubation and filled with 6 ml HBS solution, then incubated at 38 oC. 2 - 4 µl
samples were taken from the percoll washed semen suspension and added to 6 ml
iso-osmotic (300 mOsm kg-1) and hypo-osmotic (180 mOsm kg-1) HBS solution
contained in snap-cap glass vials pre-incubated at 38 oC. After an exactly determined
incubation periods (5 and 20 min), the samples were passed through a CASY1 cell
counter. At each sampling time-point, such distributions were collected from a single
iso-osmotic dilution and a single hypo-osmotic dilution using a CASY1 sample
volume setting of 200 µl and a size scale of 10 µm. The cursors were fixed at
positions 2.3 µm and 6 µm during the entire experiment to collect all representative
volume distribution fractions under different osmotic conditions.
In each sampling the data were obtained from more than 20,000 sperm cells. The
volume distributions were measured at 5 min and 20 min of incubation in both iso-
and hypo-osmotic HBS solutions. The incubation time in the measuring solutions
remained constant for all samples.
3.4.2.5 Analysis of derived volumetric parameters
The original cell-counter data were recorded for 512,000 volume channels. To
analyse and save the files, the data were formatted for 1024 effective diameter
channels. Modal value of the volume distribution curve was taken into consideration,
as the modal value is the most frequent value of a distribution, and are very stable
against the extreme-low and high values in the distribution. Moreover it was found to
be a more sensitive parameter of volume change than the mean value
Materials and Methods ___________________________________________________________________
39
(PETRUNKINA and TÖPFER-PETERSEN, 2000). The modal values of the sperm
volume distribution curves (figure 5a & 5b) were submitted to statistical analysis after
correction of such values obtained under hypo-osmotic conditions with the calculated
correction factor. The relative volume shift (RVS) was used as a measure of the
sperm volume regulation in response to hypo-osmotic stress (PETZOLDT and
ENGEL, 1994).
It was defined as: RVS = Vh5 / Vi5. Where Vh5 is the modal value of the hypo-
osmotic volume distribution of samples incubated for 5 min (figure 5), and Vi5 is the
modal value of the iso-osmotic volume distribution after 5 minutes of incubation.
When several sperm subpopulations contributed to a distribution, the values
pertaining to the largest osmotically active subpopulation were used. A cell
subpopulation was considered osmotically active if its RVS was > 1 (PETROUNKINA
et al., 2000). Regulative volume decrease (RVD) of modal values of volume
distribution curves was also used as an evaluation parameter of the functional
integrity of sperm membrane. It was defined as; RVD =RVS - Vr20, (Vr20 = Vh20 /
Vi20). Where Vh20 is the modal value of the hypo-osmotic volume distribution of
samples incubated for 20 min (figure 6), and Vi20 is the modal value of the iso-
osmotic volume distribution after 20 minutes of incubation (PETRUNKINA et al,
2001c).
Materials and Methods ___________________________________________________________________
40
(Figure 5a)
(Figure 5b)
Figure 5: Volume distribution curves of frozen-thawed bull sperm under iso-osmotic (a) and hypo-osmotic (b) conditions. Hypo-osmotic distribution curve is shifted to larger volume values compared with the iso-osmotic curve; the modal sperm volume increased from 13.50 fl under iso-osmotic (a) to 19.76 fl under hypo-osmotic conditions (b). The distribution shape is changed; the heterogeneity of response is more strongly pronounced. Vertical lines represent the cursor positions that define the sperm population selected for analysis. Particles with lower volumes (cell debris and noise) and larger volumes (agglutinated sperm) are excluded from analysis.
Materials and Methods ___________________________________________________________________
41
3.4.3 Evaluation of sperm chromatin status (mf-SCSA)
EVENSON and co-workers (1980) developed the sperm chromatin structure assay
as flowcytometric method. TEJADA et al. (1984), KOSOWER et al. (1992) and
ACEVEDO et al. (2001) modified the test, in order to make a fluorescence
microscopic evaluation possible. The protocol of ACEVEDO et al. (2001) modified by
WABERSKI and HELMUS (unpublished) was used in the present study.
3.4.3.1 Preparation of semen smears
In this experiment 90 frozen semen straws from 30 bulls (3 straws /bull) were used.
At each attempt, 12-16 fine straws (0.25 ml) were thawed in water bath at 39 oC for
10 seconds. The contents were transferred to a graduated centrifuge tubes and filled
up to 2 ml with sodium citrate buffer 2.9 % (6.8 pH), then washed two times by
centrifugation at 2700 g (3500 rpm) for 10 minutes at room temp (20 oC). The
supernatants were sucked off by means of water-operated vacuum pump and the
pellets were resuspended each in 2 ml sodium citrate buffer 2.9 % and vortexed to
ensure proper pellet disruption. After the second centrifugation, the supernatants
were sucked off and the sperm pellets were separately resuspended each in100 µl
citrate buffer. Two thick semen smears were made by placing 50 µl semen
suspension on a special glass slide (Superfrost® plus; company Roth, KARLSRUHE)
marked with a solvent-free pin (acid- and alcohol proof). The smears were left at
room temperature for approximately 10 minutes for air-drying. The further steps of
this protocol were accomplished either at the same day or on the following days.
3.4.3.2 Decondensation of sperm chromatin
The procedures for preparation of the solutions used for decondensation and
denaturation of sperm chromatin were carried out under an Outlet (vent) using
protective masks and gloves. Decondensation solution must be used within 24 h. For
each slide, 2 ml decondensation solution was needed. Per passage, 16 smears could
be stained (i.e. 32 ml decondensation solution was needed). For preparation of 32 ml
solution, 0.0247 gm 1.4-Dithiothreit (DTT) was weighed out on a laboratory balance
Materials and Methods ___________________________________________________________________
42
and transferred to a beaker containing 32 ml sodium citrate 2.9 % buffer
(corresponds to 5 mM DTT). The next step, 1 mM Dimethylsulfoxide (DMSO) was
diluted with 1.75 mM distilled water. Into a separate test tube 2.272 ml DMSO were
added with a syringe (correspondence to 1 mM DMSO; 71 µl / ml) and mixed with
1008 µl bi-distilled water (correspondence to 1.75 mM water; 31.5 µl / ml). After
accomplish of heat-produced reaction and clear cooling of the test tube, its contents
were added to the beaker containing DTT solution. The slides were kept horizontally
on a test tube stand under the Outlet (Vent). Each slide was covered with 2 ml of the
work solution (DTT / DMSO) and allowed to stand for 30 minutes. Shortly before
ending of the decondensation time, a washing bottle with sodium citrate buffer 2.9 %
(6.8 pH), a jar with the same buffer and a beaker were being made available. The
remained denaturing solution on the slides were poured off in turn over the beaker,
rinsed with the washing bottle and placed for eight minutes in a rinsing jar. Afterwards
the slides were taken out, dried on the back with cellulose and placed as
perpendicularly as possible on an absorbent material (cellulose), in air for
approximately 10 minutes.
3.4.3.3 Acid denaturation
The air-dried semen smears were put in a jar containing 60 ml Carnoy’s Solution (20
ml acetic acid + 40 Methanol, pH value 2) for 100 minutes for acid-denaturation. 15
minutes before the end of denaturation time, a staining jar, and a rinsing jar were put
in a refrigerator. Upon completion of the denaturation time, the slides were taken out
from the jar and wiped with cellulose and then quickly dried in air.
3.4.3.4 Staining with acridin orange (AO)
The prepared semen smears were placed in a pre-cooled staining jar containing AO
staining solution, which consisted of 40 ml citric acid solution + 2.5 ml cooled di-
sodium hydrogen phosphate solution + 10 ml AO stock solution (cooled, darkly
stored) and stored in a refrigerator for 20 min. After staining, the slides were washed
gently with a pre-cooled sod citrate buffer 2.9 % then placed in a washing jar
Materials and Methods ___________________________________________________________________
43
containing the same buffer. 10 min later the smears were taken out from the washing
jar, allowed to dry and maintained in a refrigerator until viewing.
3.4.3.5 Evaluation of the stained smears
Evaluation of the stained semen smears was carried out in a darkened room by
means of fluorescence microscope using a blue laser (490 nm excitation filter, and
520-nm barrier filter), phase 2 and 200x magnification with a 20 objective lens.
Thereby, the wavelength 530 nm light emits green (double strand DNA) and 640 nm
light emits red (single strand DNA). A digital camera (Olympus DP 50) was mounted
onto the fluorescence microscope and coupled with a computer that processes and
downloads the digital image through a program called Software analysis® 3.0 (Soft
Imaging System GmbH, MÜNSTER). A series of fields per slide were photographed
and saved for later evaluation. For each replicate, AO stained spermatozoa were
assessed simultaneously in more than 200 spermatozoa in 10 or more individual
fields. The fluorescent characteristics of each cell were noted as green (chromatin
stable, double-stranded, acid resistant DNA), red-orange (chromatin unstable, single
stranded, denatured DNA), half green-half red (partially denatured DNA), pink or
yellow (partially denatured DNA) as shown in figure 6.
Materials and Methods ___________________________________________________________________
44
Figure 6: Computer-assisted evaluation of sperm chromatin status with ANALYSIS® 3.0 program. a) mf-SCSA-Fluorescence-live picture, b) Detected sample (digital overlay).
3.4.4 Oviductal explant assay (OEA)
The cows and heifers included in this experiment were clinically healthy but of
unknown previous reproductive history.
3.4.4.1 Preparation of oviductal explants
Oviducts including isthmus, ampulla, infundibulum, fimbria, small part of the utero-
tubal junction and mesosalpinx were collected from both cows and mature heifers at
the local slaughterhouse in Hannover city. The oviducts were collected within 20-30
min of the animal’s death. The uterus and ovaries were examined for anomalies and
pathological lesions as well as for pregnancy before disposal of the oviducts.
Each oviduct was thoroughly washed with sterile PBS and placed in 100 ml of PBS
(pH 7.4) then transported on ice to the laboratory. Upon arrival, the oviducts were
thoroughly washed with PBS and then dissected free of the surrounding tissues
(mesosalpinx) and straightened as much as possible. The ampullary and isthmic
a b
Materials and Methods ___________________________________________________________________
45
segments were cut into 2-3 pieces in large Petri-dish containing PBS. Each piece
was taken with a watchmaker’s forceps (tweezers) and held from the narrow end
over a small Petri-dish containing few drops of sperm-TALP medium and gently
squeezed along the outside toward the wide end with another watchmaker’s forceps
to expel epithelium. At every attempt, both oviducts (right and left) from 3-4 cows /
heifers were pooled to avoid the individual cow effect as well as the local hormonal
effect. The expelled epithelial tissue sheets were disaggregated into small pieces by
one passage through a 25-gauge needle attached to 1-ml insulin syringe and
transferred to test tube containing 5 ml sperm-TALP, then allowed to stand for 10
min. After initial sedimentation, the supernatant was removed and 5 ml of fresh
sperm-TALP was added to the pellet. The same volume was removed again after 10
min (second sedimentation) and the Oviduct Epithelial Cells (OEC) sheets were
resuspended in 0.5 ml sperm-TALP and incubated at 39 oC in a humidified
atmosphere containing 5 % CO2. Within 30 min of disaggregation, the clumps of
epithelial cells formed everted vesicles with apical surfaces facing outward,
henceforth referred to as ´´explants´´ (figure 7a).
Figure 7: a) Bovine sperm bound to oviductal epithelial Explants (Phase contrast microscope 200x); b) Scanning micrograph (5000x), bovine sperm bound to the cilia of bovine Oviduct epithelial cells.
b a
Materials and Methods ___________________________________________________________________
46
3.4.4.2 Preparation of semen samples
In this experiment 90 ejaculates from 30 bulls (3 ejaculates / bull) were used at 35
attempt days. At each attempt day, 3-4 straws from different bulls were thawed and
examined subjectively for motility, then passed through discontinuous percoll
gradients as described in m-HOST experiment. Aliquots of the recovered
spermatozoa were suspended separately in 0.3-0.5 ml warm sperm-TALP and
assessed again for motility (motility of washed spermatozoa) on a microscope stage
heated at 38 oC and sperm cell concentration using a haemocytometer.
3.4.4.3 Determination of sperm cell concentration
Estimation of the sperm cell concentration was carried out by means of Thoma cell
counting chamber. With a micropipette, 20 µl thawed semen was taken and diluted in
480 µl 10 % sodium chloride (dilution rate = 1:25). A coverslip was placed on the
haemocytometer counting slide after wetting supports with saliva to tightly hold the
coverslip while loading the sperm. 10 -15 µl of the diluted sperm was allowed to flow
under the cover slip on each side of the haemocytometer. After 5 minutes the slide
was viewed under a phase contrast microscope using 40x magnifications.
On each side of the haemocytometer, the spermatozoa were counted in 5 large
squares (4 diagonal and one at the corner). The calculation of the sperm cells density
in million per µl was made according to the following formula:
Number of spermatozoa counted in 10 squares The number of sperm /1 µl =
Counted surface area x chamber depth x dilution rate
The haemocytometer chamber is 0.1 mm in depth and the 25 large squares
represent an area of 1 mm2. The volume above the 25 squares shown is 0.1 µl. Only
10 squares were counted and the dilution rate was 1:25, so a factor of 625 was
calculated, by which the sum of the counted spermatozoa was multiplied. The result
corresponded to the concentration of the sperm cells per µl solution. The sperm cell
concentration was adjusted to 5 x 106 / ml.
Materials and Methods ___________________________________________________________________
47
3.4.4.4 Co-incubation of spermatozoa with oviductal explants
Both the explants and the prepared semen samples were equilibrated for 10 min at
39 oC in a humidified atmosphere containing 5 % CO2. Afterwards, 10 µl aliquot was
taken from the dense layer of explants and transferred to 50 µl droplet of sperm-
TALP in a small Petri dish, then 20 µl semen suspension was added to the droplet
and gently mixed, so that the final droplet volume was 80 µl, and the final sperm cell
concentration was 1.25 x 106 sperm /ml. After 15 min of co-incubation in CO2
incubator, the explants were washed free of unbound, loosely attached sperm by
drawing them up into a 100-µl micropipette and transferring them into fresh 80 µl
sperm-TALP droplets. The washing process was repeated three times to assure that
all unbound sperm were removed. The explants with bound sperm were then
transferred to pre-warmed slide supported by silicon grease and covered with pre-
warmed cover slips, then pressed gently for fixation.
3.4.4.5 Video-microscopy and image analysis
The prepared slide was transferred with the cover slip directed downward to a warm
stage (38 oC) inverted microscope (IM35; company Zeiss, JENA / GERMANY)
equipped with video camera (Kappa, CF 8/1) also coupled with video recorder (SLV-
E 720, VHS; company Sony, Japan) and monitor (WV-3M 1400; Panasonic, JAPAN).
Explants on each slide were viewed under 256x magnifications (32 x 8). 2 slides per
ejaculate were made and in each slide, 6 fragments (sections) of about 2-3 explants
(1-2 fragments / explant) were videotaped. All attempts were successively
accomplished and taken up on videocassette, so that the photographs could be
evaluated to a later time. Beside the bound sperm cells on each attempt day a scale
was videotaped in the respective magnification. Videotaping was completed within 12
min for each slide. For analysis, the videotapes were reviewed to count the number
of spermatozoa bound to the side of the oviduct explants facing the camera. For
counting out the bound sperm, a foil was put over the image plane of the monitor,
then the bound spermatozoa were marked with a water-soluble marker and these
markings were counted and documented.
Materials and Methods ___________________________________________________________________
48
3.4.4.6 Estimation the surface area of the explant
The surface areas of the videotaped explant and its fragments were estimated with
the help of an image analysis, computer-assisted, surface area measuring program
"Aida" (Mika medical GmbH image analysis version 2.0; Copyright 1992; Rosenheim,
GERMANY). The computer was coupled with a monitor and video recorder. The
scaling for the respective enlargement had to be stored and the computer program
was scaled firstly before the computer could accomplish the computations. The
videotaped explants were stored separately in the fixed image in order to be able to
mark their contour with the mouse on the monitor.
3.4.4.7 Determination of the binding index (BI)
The number of spermatozoa bound to 0.01 mm2 explant’s surface was used as a
parameter of sperm- oviduct binding capability and called binding index (BI). The
calculating BI was adapted after PETRUNKINA et al. (2001b).
The surface areas of 36 fragments per bull (12 fragments / ejaculate and 3 ejaculates
/ bull) and their bound sperm numbers were submitted to the Mean procedure of SAS
to determine the BI for each ejaculate according to the formula:
� N1+N2+………………N12 BI =
� S1+S2…………………S12
Where N1-12 = the number of bound spermatozoa / fragment and S1-12 = the surface
areas of the explant`s fragments. The BI for each bull was calculated as the mean
value of the binding indices of the three ejaculates.
3.4.5 In vitro fertilization (IVF)
Two experiments were carried out at Institute of animal breeding in Mariensee, the
federal research institute for agriculture (FAL). The first experiment was carried out in
the period between April / 2002 and September / 2002 to investigate the relationship
between percentage of spermatozoa with unstable chromatin and the IVF results
(cleavage- and blastocyst-rate). The second experiment was carried out between
February / 2003 and May / 2003 to investigate the relationship between the ability of
Materials and Methods ___________________________________________________________________
49
spermatozoa to bind to oviductal epithelium and in vitro fertility (cleavage- and
blastocyst-rate). The procedure that described by WRENZYCKI (1995) with some
modifications was used.
In the first experiment, 16 straws from 4 different bulls (4 straws per bull) and 5
straws from one bull, altogether 21 straws from five bulls were examined on 7
attempt days. On each attempt day, three straws from three different bulls were
examined. Per straw approximately 55-60 oocytes were used, so that 1217 oocytes
were used in the entire experiment. The 5 bulls were divided into 2 groups, the first
group include two bulls with relatively low mf-SCSA values (3.6 ± 0.7), and the other
three bulls (group II) had relatively high mf-SCSA values (7.6 ± 0.4) (table 5).
In the second experiment 20 straws from four bulls (5 straws per bull) and 12 straws
from 2 bulls (6 straws per bulls), altogether 32 straws of the six bulls were examined
on 10 attempt days. On each attempt day 3-4 straws from different bulls were
examined. Per straw approximately 50-60 oocytes were used, so that 1899 oocytes
were used in the entire experiment. The six bulls in this experiment were divided into
two groups. The first group included three bulls with relatively high binding indexes
(19.9±2.4) and the second group include bulls with relatively low binding indices
(10.4±0.5) as shown in table 8.
3.4.5.1 Collection of ovaries
Ovaries were recovered 20 min after slaughtering from cows and heifers at the
slaughterhouse in LÜBBECKE city. The animals were mostly of Holstein origin,
whose age and medical history were unknown. No selection with respect to the stage
of oestrous cycle was done. However, ovaries from cows with uterine pathology such
as pyometra, or ovarian cysts were not collected. Ovaries were collected on fat
(mesentery) in an insulated flask (thermos bottle) and being transported to the
laboratory within two hours. Upon arrival, the ovaries were washed 2-3 times with
warm (30 oC) PBS medium before the slicing began.
Materials and Methods ___________________________________________________________________
50
3.4.5.2 Recovery of oocytes
The oocytes were recovered from the ovaries by the slicing method. The slicing units
consist of 6-8 razor blades (0.15 mm, Romi, Solingen, GERMANY) which joined
together in a metal skeleton. The slicing device cuts the surface of the ovaries in
various dimensions. Ovaries were held in large Petri dish and fixed with artery
forceps in PBS medium supplemented with 2 IU heparin (0.0056 g / 500 ml) and 0.1
% BSA. The slicing was made in different dimensions with about 3 mm depth.
Following slicing, the resulting fluid was passed through a fine sieve into a glass
beaker and allowed to stand ~15 min for sedimentation of cumulus oocyte
complexes. The supernatant was removed by means of a water-operated vacuum
pump and the sediment re-suspended in about 100 ml PBS (with heparin) was
transferred to 15 ml centrifuge tubes (Greiner, NÜRTINGEN, GERMANY) and then
the sediment was removed and diluted with fresh PBS (with heparin) medium in 60
mm plastic dishes (Greiner GmbH, NÜRTINGEN, GERMANY) before being viewed
under a stereomicroscope. Only class I, i.e. oocytes with a homogeneous evenly
granulated cytoplasm possessing at least three layers of compact cumulus cells and
class II, i.e. oocytes with fewer than three layers of cumulus cells or partially denuded
but also with a homogeneous evenly granulated cytoplasm were selected and
transferred to warm collection medium (TCM-air) in small Petri dish on a warm (38 oC) plate. The oocytes were transferred to the maturation medium. Oocytes with
degenerated cytoplasm or surrounded by expanded, degenerated, dark looking
cumulus cells, were not used in the present study (figure 8).
Materials and Methods ___________________________________________________________________
51
Figure 8: In vitro bovine cumulus oocyte complex, classes I and II.
3.4.5.3 In vitro maturation (IVM)
On the day of use, TCM199 medium was supplemented with 10 % BSA and pyruvate
(2.2 mg / 100 ml) to produce washing medium (TCM-pure). A portion of this medium
(975 µl) was supplemented with 25 µl Suigonan® (One dose Suigonan
® consists of
200 IU hCG and 400 IU eCG, Intervet, TÖNISVORST, GERMANY) to serve as
maturation medium. In a medium size culture dishes, the wash drops were prepared
at a rate of 12 drops (100 µl) per dish then covered with silicon oil (Serva,
HEIDELBERG, GERMANY). For maturation, four 100-µl droplets were prepared in
35 mm sterile polystyrene culture dishes (Greiner GmbH, NÜRTINGEN, GERMANY),
then covered with silicone oil and equilibrated in the same culture environment for
one h .The immature oocytes were washed three times in washing drops before
being transferred in groups of 20-25 to the maturation drops. Equilibration and
incubation were carried out at 39 oC in high humidity atmosphere and 5 % CO2 in air
for 23-24 h.
Class I Class II
Materials and Methods ___________________________________________________________________
52
3.4.5.4 In vitro fertilization (IVF)
Modifications of Tyrode Albumin Lactate Pyruvate (TALP) medium after (PARRISH et
al., 1988) were used. Sperm-TALP was employed for swim-up separation of the
motile fraction of semen and subsequent washing of sperm. It was supplemented
with pyruvate and BSA (A-9647 fraction V, Sigma) on the day of use. The other
modification, fert-TALP was used for washing of the IVM oocytes before they were
placed into the fertilization drops made from fert-TALP medium. This medium was
supplemented with gentamicinsulfat, sodium pyruvate and BSA on the day of use.
The IVF media were prepared in double distilled water (Ampuwa®, Fresenius AG)
and the pH was adjusted to 7.4 then stored at 4 oC after passing through a 0.22 µm
in Ø cellulose sterile filter. The fertilization medium was prepared freshly by
supplementing fert-TALP with the capacitation inducing agents consisting of
hypotaurine, epinephrine and heparin. Both washing and fertilization media were
equilibrated in the culture environment for one h prior to insemination. The in vitro
matured oocytes were washed three times in washing drops under oil and transferred
in groups of 20-25 oocytes to the fertilization droplets. The oocytes were then
returned to the incubator for at least 30 min until sperm preparation was
accomplished.
3.4.5.5 Preparation of spermatozoa and fertilization
Semen was prepared as described by PARRISH et al. (1988). In each attempt three
straws from three different bulls were group thawed in water bath at 38 oC for 1 min.
For swim-up separation of the motile fraction, the content of each straw (0.25 ml) was
layered under 1 ml sperm-TALP supplemented with BSA (A-9647 fraction V, Sigma)
and pyruvate in sterile glass held at an angle of 45o The motility of the sperm after
thawing was determined under a phase contrast microscope (200x). After one h of
incubation at 39 oC under 5 % CO2 in air, 850 µl from the top of the medium was
pipette and transferred into a sterile centrifuge tube. Following the addition of 5 ml
sperm-TALP medium, the swim-up separated sperm were centrifuged at 350 g (1200
RPM) at 25 oC for 10 min. The sperm pellets were resuspended each in fresh 5 ml of
Materials and Methods ___________________________________________________________________
53
sperm-TALP medium and centrifuged again. The final sperm pellets were
resuspended each to ~200 µl with fert-TALP and incubated for 15 min at 39 oC under
5 % CO2 in air for capacitation. During this time sperm concentration was determined
using a counter slide (Thoma; Superior, Omnilab, GEHRDEN, GERMANY). The
concentration was adjusted to 12.5 million sperm per ml using the fertilization
medium for dilution. 2 µl aliquots sperm suspension were transferred to each 100 µl
fertilization droplet containing ~20 oocytes to give a final sperm concentration of 0.25
million sperm per ml (suboptimal sperm concentration to be able to differentiates
among bulls).
3.4.5.6 Removal of cumulus cells
Fertilized oocytes were denuded from the cumulus cells by vortexing (1200 / min) for
4 min in collection medium (TCM-air) followed by gentle pipetting and collection the
denuded ova under a stereomicroscope.
3.4.5.7 In vitro culture of embryos (IVC)
On the day of use, the stock solution of synthetic oviductal fluid (SOF medium) was
supplemented with Na-Pyruvate, glutamine, Gentamycin, non-essential amino acids,
essential amino acids and polyvinyl alcohol (see appendix). This medium was used
as wash and culture medium. Prior to use, the wash and culture dishes were
equilibrated in the culture environment for one h. About 18 h following fertilization,
presumptive zygotes were denuded of cumulus cells, washed three times in 80 µl
droplets of washing medium and then transferred in groups of 6-8 zygotes into 30 µl
of culture medium. Zygotes were cultured under silicone oil in 5 % CO2, 5 % O2 and
90 % N2 (Air Product, HATTINGEN, GERMANY) in a humidified atmosphere in
Modular incubator (ICN Biomedical, Inc., Aurora, No. 615300, OHIO, USA) at 39 oC
for 8 days. The culture medium was not replaced during the culture period. Cleavage
rate was evaluated under a stereomicroscope at 45× magnification on day 3 by
counting the 2 to 8 cell embryos and referred to the whole of the cultivated embryos
also the blastocyst rate was determined on day 8. The embryonic stages were
Materials and Methods ___________________________________________________________________
54
assessed under a stereomicroscope at 45x magnification after denudation and given
below according to LINDNER and WRIGHT (1983) as shown in figure 9.
a) In vitro derived bovine 2-4-cell embryo b) In vitro derived bovine 4-8-cell embryo c) In vitro derived bovine 8-16 cell embryo d) In vitro derived bovine morula e) In vitro derived bovine expanded blastocyst f) In vitro derived bovine hatched blastocyst
Figure 9: Different embryonic stages after LINDNER and WRIGHT (1983).
b a
d c
f e
Materials and Methods ___________________________________________________________________
55
3.5 STASTICAL ANALYSIS
The computation and the diagrams of the present study were accomplished using the
statistics package SAS/ STAT (SAS institute Inc., version V8.3 for Windows, Cary,
North Carolina, USA) as well as the Excel software (Microsoft office XP, Inc., USA).
The data acquisition and organization were carried out with the data base program
(dbase for Windows, version 3.0). Data were not transformed because they passed
tests of normality and homogeneity of variances.
3.5.1 Analysis of volumetric parameters
The data recovered from Latex particles calibration were submitted to the linear
regression (REG procedure) of SAS to calculate the correction factor. The calculated
correction factors were 1.16 for modal values and 1.11 for the mean values of the
sperm cell volume under hypo-osmotic conditions.
The corrected CASY data were analysed by the general linear models procedure of
SAS (GLM, Least Squares Means). Means are reported as least square mean (LSM
± SD) unless stated otherwise. Comparisons were made within the volume
distributions obtained from the replicate ejaculates and among individual bulls.
3.5.2 Analysis of the data of mf-SCSA
The evaluation was done by means of the statistics procedure MEANS of the SAS
package. For the question, in any degree the mf-SCSA value was connected with the
other spermatological parameters, the Pearson's coefficient of correlation was
computed with the SAS procedure CORR. In addition, for the classical spermatology
parameters, a stepwise multiple regression analysis was accomplished, in order to
find out whether one of the variables had a prognostic value for mf-SCSA.
3.5.3 Analysis of the data of oviductal explants assay (OEA)
The average values and the standard deviations of the binding index were separately
calculated for each individual bull using the procedure MEANS. These values served
Materials and Methods ___________________________________________________________________
56
as design fundamentals for further analyses of a total average value over all bulls
and for the comparison among individual bulls. For comparison between bulls the 2-
fatorial analyses of variance (ANOVA) were accomplished using general linear model
(GLM procedure). For determination of the relation between binding index and other
spermatological parameters, the correlation analysis was accomplished after
Pearson by calculation of the correlation coefficient (r) and the associated probability
of mistake (p value) using the procedure CORR of SAS.
3.5.4 Analysis of the IVF data
In the first experiment, which performed to investigate the relationship between
sperm chromatin status and the IVF results (cleavage and blastocyst rate), the
normal distribution of the results was examined. Since no normal distribution was
present, no correlation was computed. Subsequently, the samples were divided with
low and high mf-SCSA values into two groups and compared with each other using
Wilcoxon two-sample test and in t-test.
Concerning the second IVF experiment that performed to study the relation between
sperm oviduct binding ability and fertility In vitro the bulls were classified into two
groups according to their binding indices, one with relatively high BI and the other
with relatively low BI. The data from six bulls included in this experiment were
submitted to the GLM procedure, least square means, NPAR1WAY procedure
(ANOVA) and Wilcoxon two-sample test to differentiate between two groups of bulls.
3.5.5 Significance levels for the probability of mistake
For the entire study applies the value of p � 0.05 as a significant limit value for the
probability of null hypothesis. A further level of P � 0.001 was used, which indicates
the limit value of a high-significant probability of mistake. All data represented as
mean value ± standard deviation (SD).
Results ___________________________________________________________________
57
4 RESULTS
4.1 STANDARD SPERMATOLOGICAL PARAMETERS
4.1.1 Motility parameters
4.1.1.1 Post-thawing subjective motility
The descriptive statistics of the sperm motility parameters for the 30 bulls involved in
the present study are listed in table 1. The motility of frozen-thawed semen was
evaluated immediately after thawing. Forward- and local-motile as well as non-motile
spermatozoa were estimated to the nearest 5 %. The forward motility % ranged from
40 ± 18.1 % to 75 ± 5 %. The overall mean percentage of forward motile
spermatozoa was 60.4 ± 8.2 % (mean ± SD). The most of bulls (26 bulls) recorded
values higher than 50 % (the minimum required value), while only 4 bulls had values
lower than 50 % as shown in figure 10.
Figure 10: Post-thawing subjective progressive forward motility %. (90 ejaculates from 30 bulls; 3 ejaculates / bull).
-
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Bull No.
Pro
gres
sive
for
war
d m
otili
ty (%
)
Results ___________________________________________________________________
58
4.1.1.2 Post thawing motility using cell motion analyser (CMA)
The overall mean percentage of forward motile spermatozoa was 59.2 ± 8.4 % (mean
± SD), with a maximum value of 72.8 % and a minimum one of 34.3 %. It was not
significantly different compared with the subjectively estimated value (60.4 ± 8.2 %),
as shown in table 1. Only 4 bulls had recorded forward motility values higher than 50
% (figure.11). The average path-, curvilinear- and straight-line velocities of
spermatozoa were 62.75 ± 3.9, 112 ± 8.1 and 55.9 ± 4.7 µm / sec respectively (table
1).
-
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bull No.
Pro
gres
sive
for
war
d m
otili
ty (%
)
Figure 11: Post-thawing progressive forward motility %, estimated with CMA (90 ejaculates from 30 bulls; 3 ejaculates / bull).
Results ___________________________________________________________________
59
4.1.1.3 Motility of percoll selected spermatozoa
Immediately after centrifugation of the frozen thawed semen samples on
discontinuous percoll gradients, the sperm pellets were resuspended in 200-300 ml
warm sperm-TALP and incubated at 39 oC for 5 minutes in CO2 incubator, then
subjectively examined for motility. A highly significant (P < 0.0001) difference was
obtained between the forward motility percentage before and after percoll washing.
There was a rise from 60.4 ± 8.2 % before washing to 74.5 ± 7.5 % (mean ± SD)
after washing, with a range from 55.0 % to 88.4 %. All bulls had recorded values
higher than 50 % (the minimum required threshold value of forward motility %) as
shown in figure.12.
-
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bull No.
Pro
gres
sive
for
war
d m
otili
ty (%
)
Figure 12: Post-thawing subjective progressive forward motility % of percoll selected spermatozoa (90 ejaculates from 30 bulls; 3 ejaculates / bull).
Results ___________________________________________________________________
60
Item Mean SD Minimum Maximum
Subj-f 60.4 8.2 40.0 75.0
Subj-l 8.9 2.5 5.0 10.0
Subj-i 30.7 10.6 15.0 85.0
Perc-f 74.5 7.5 55.0 88.4
Perc-l 9.4 2.3 5.0 15.0
Perc-i 16.1 7.8 3.4 35.0
CMA-f 59.2 8.4 34.3 72.8
CMA-l 8.1 2.9 1.0 16.0
CMA-i 32.7 9.4 17.1 61.1
VAP 62.75 3.9 47.2 62.4
VCL 112.0 8.1 87.1 125.4
VSL 55.9 4.7 33.8 65.5
Table 1: Mean, minimum and maximum values of sperm motility parameters (n = 90 ejaculates from 30 bulls; 3 ejaculates / bull).
Forward motile Spermatozoa (roughly estimated %) Local-motile spermatozoa (roughly estimated %) Immotile spermatozoa (roughly estimated %) Forward motile % of percoll washed spermatozoa (roughly. estimated) Local-motile % of percoll washed spermatozoa (Roughly estimated) Immotile % of percoll washed spermatozoa (roughly estimated) Forward motile Spermatozoa (CMA-determined %) Local-motile spermatozoa (CMA-determined %) Immotile spermatozoa (CMA-determined %) Average path velocity of spermatozoa (µm/sec.) Curvilinear velocity of spermatozoa (µm/sec.) Straight-line velocity of spermatozoa (µm/s) Cell motion analyzer Standard deviation
Subj-f: Subj-l: Subj-i: Perc-f: Perc-l: Perc-i: CMA-f: CMA-l: CMA-i: VAP: VCL: VSL: CMA: SD:
Results ___________________________________________________________________
61
4.1.2 Morphological abnormalities of spermatozoa
Spermatozoa with abnormal morphology were classified into two categories.
Category 1) HEAD: including primary- and secondary head abnormalities as well as
acrosomal abnormalities and category 2) MAS (morphologically altered
spermatozoa): including head abnormalities and other abnormalities such as
midpiece- and tail abnormalities.
The overall mean percentage of head abnormalities was 30.4 ± 6.6 % (mean ± SD)
with a maximum value of 43 % and a minimum one of 21.2 %. Concerning
morphologically altered spermatozoa (MAS), the overall mean was 37.4 ± 7.6 %
(mean ± SD) and ranged from; 25.4 % to 51.9 % (table 2). All bulls had recorded
values less than 50 % except 2 bulls (figure 13).
Item SD Minimum Maximum
Prim-head 0.8 0.7 0.0 4.0
Acrosome 29.6 7.9 15.6 38.4
Head 30,4 6,6 21,2 43,0
Others 6,6 4,1 1,0 26,0
MAS 37.4 7,6 25,4 51.9
Alive 65,6 10,8 26,1 80,9
Dead 34.5 10.8 19.1 73.9
Table 2: Morphological parameters and viability of frozen thawed semen (90 ejaculates from 30 bulls; 3 ejaculates per bull).
x
Primary sperm head abnormalities % Acrosomal abnormalities % Primary head, secondary head and acrosomal abnormalities Sperm tail and midpiece abnormalities Morphologically altered spermatozoa Alive sperm % Dead sperm %
Prim-head: Acrosome: Head: Others: MAS Alive: Dead:
Results ___________________________________________________________________
62
-
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bull No.
Tota
l sp
erm
abn
orm
aliti
es (%
)
Figure 13: Total percentage of morphologically altered spermatozoa (MAS) (90 ejaculates from 30 bulls; 3 ejaculates / bull).
4.1.3 Relationship between IVF results and standard
spermatological parameters
When the data of the bulls that were included in the two IVF experiments (n=11) were
submitted to statistical analysis, no significant correlations existed between cleavage
rate and any of the standard spermatological parameters. However, significant (P �
0.05) Correlations were recorded between blastocyst rate and alive sperm %, sperm
head abnormalities and total sperm cell abnormalities (table 3).
Results ___________________________________________________________________
63
Item Cleavage Rate Blastocyst Rate
r -0.03 0.46 RVS
p 0.94 0.18
r -0.13 0.02 RVD
p 0.70 0.94
r -0.43 0.06 Subj-f
p 0.21 0.86
r -0.36 -0.36 VSL
p 0.29 0.29
r -0.02 -0.02 VAP
p 0.93 0.93
r -0.20 -0.56 VCL
p 0.09 0.09
r 0.49 0.65 Alive
p 0.14 0.04
r -0.37 -0.67 Head
p 0.28 0.03
r -0.38 -0.68 MAS
p 0.26 0.02
Table 3: Pearson’s correlation coefficients and levels of significance between IVF
results and some spermatological parameters (n = 11 ejaculate from 11 Bulls). r = correlation coefficient, p = degree of probability.
Modal value of relative volume shift of spermatozoa Modal value of regulative volume decrease of spermatozoa Forward motile Spermatozoa (roughly estimated %) Straight-line velocity of spermatozoa (µm / sec) Average path velocity of spermatozoa (µm / sec) Curvilinear velocity of spermatozoa (µm / sec) Alive sperm (%) Sperm head abnormalities including acrosomal abnormalities (%) Morphologically altered spermatozoa (%)
RVS RVD Subj-f VSL VAP VCL Alive HEAD MAS
Results ___________________________________________________________________
64
4.2 ADVANCED SPERMATOLOGICAL PARAMETERS
4.2.1 Viability assessment using LIVE/DEAD® Sperm Viability Kit
The overall mean alive sperm percentage was 65.5 ± 10.8 % (mean ± SD) with a
minimum value of 26.1 % and a maximum one of 80.9 % (table 2).
Concerning the differences among individual bulls, more than 83 % of bulls (n = 25)
showed values between 54.8 % (mean - SD) and 76.32 % (mean + SD), while only 3
bulls showed values less than 54.8 % as shown in figure 14.
-
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bull No.
Aliv
e s
perm
(%
)
Figure 14: Alive sperm % as determined using LIVE/DEAD® Sperm viability kit. (90 ejaculates from 30 bulls; 3 ejaculates / bull).
4.2.2 Modified hypo-osmotic swelling test (m-HOST)
The descriptive statistics of the sperm cell volumetric parameters are illustrated in
table 4. The overall mean of the modal values of sperm cell volume distribution under
iso-osmotic conditions remained nearly constant along the entire incubation period
Results ___________________________________________________________________
65
(13.4 ± 3.6 fl at 5 min vs. 13.8 ± 3.5 fl at 20 min), but increased to 21.3 ± 4.8 fl (mean
± SD) under hypo-osmotic conditions after 5 min of incubation at 38 oC, then
decreased to 18.8 ± 4.9 fl (mean ± SD) after 20 min of incubation under hypo-osmotic
conditions.
Table 4: Sperm cell volumetric parameters (90 ejaculates from 30 bulls).
Item SD Minimum Maximum Vi5m 32.5 2.4 26.4 fl 41.3 fl Vh5m 39.1 3.3 32.1 fl 50.1 fl Vi20m 32.8 3.1 23.3 fl 41.7 fl Vh20m 38.4 3.6 31.2 fl 49.8 fl Vr5m 1.2 0.1 1.1 1.5 Vr20m 1.2 0.1 0.1 1.6 RVDm -0.1 0.2 -0.4 0.4
Vi5 13.4 3.6 9.7 fl 33.0 fl Vh5 21.3 4.8 13.1 fl 31.9 fl Vi20 13.8 3.5 10.2 fl 29.5 fl Vh20 18.8 4.9 11.7 fl 32.2 fl RVS 1.7 0.5 0.9 2.7 Vr20 1.4 0.4 0.7 2.8 RVD 0.3 0.6 -1.2 1.6
Mean value of sperm volume under iso-osmotic conditions at 5min (fl) Mean value of sperm volume under hypo-osmotic condition at 5 min (fl) Mean sperm volume under iso-osmotic condition at 20 min (fl) Mean value of sperm volume under hypo-osmotic condition at 20 min (fl) Relative shift of mean value of sperm volume after 5 min Relative shift of mean value of sperm volume after 20 min Regulatory volume decrease (mean value) Modal value of sperm volume under iso-osmotic conditions at 5min (fl) Modal value of sperm volume under hypo-osmotic condition at 5 min (fl) Modal value of sperm volume under iso-osmotic condition at 20 min (fl) Modal value of sperm volume under hypo-osmotic condition at 20 min (fl) Relative volume shift (modal value) Relative shift of modal sperm volume after 20 min Regulatory volume decrease (modal value) Femtoliter
Vi5m Vh5m Vi20m Vh20m Vr5m Vr20m RVDm Vi5 Vh5 Vi20 Vh20 RVS Vr20 RVD fl
x
Results ___________________________________________________________________
66
4.2.2.1 Relative volume shift (RVS)
The overall mean value of relative sperm volume increase was 1.7 ± 0 (mean ± SD)
with a minimum value of 0.9 and a maximum one of 2.7. The most of bulls recorded
values between 1.4 and 2. Only three bulls had shown values more than 2 (mean +
SD), while six bulls showed values less than 1.4 (mean - SD). Additionally, clear
significant differences in RVS among bulls were observed as shown in figure 15.
c
c
abd
cd c
acd
acd
abcd acd
abcd
abcd
acd
acd
ab b
ab
abcd
abd
acd acd
cd
abcd
c acd
acd acd
abcd abd
abcd
abcd
0,0
0,5
1,0
1,5
2,0
2,5
3,0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
BULL No.
Rel
ativ
e vo
lum
e sh
ift (R
VS
)
Figure 15: Relative shift of modal sperm cell volume (n = 90 ejaculates from 30 bulls; 3 ejaculates per bull). Bulls with different letters have significantly (p � 0.05) different values.
Results ___________________________________________________________________
67
4.2.2.2 Regulatory volume decrease (RVD)
Concerning the regulative decrease of modal values of sperm volume distribution
curve (RVD), significant differences were reported among bulls (figure 16). The
overall mean was 0.3 ± 0.6 (mean ± SD) with a minimum value of -1.2 and a
maximum value of +1.6. Two bulls recorded values more than 0.7 (mean + one SD),
and only three bulls had values less than 0.2 (mean – one SD), while the rest of bulls
(18 bulls) recorded values between -0.2 and +0.7 (figure 16).
cd
ac ac
ac
ac
abd
cd
cd
ac
c
cd
abd
ab
ab ab
b
abd
cd
ac
cd
abc
abc
c
c
acd abc
abd
ab ab
ab
-1,0
-0,5
-
0,5
1,0
1,5
2,0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bull No.
Reg
ulat
ory
Vol
ume
Dec
reas
e (R
VD
)
Figure 16: Regulatory volume decrease (RVD). (n = 90 ejaculates from 30 bulls; 3
ejaculates per bull). Bulls with different letters have significantly (p � 0.05) different values.
Results ___________________________________________________________________
68
4.2.3 Modified florescence microscopical SCSA (mf-SCSA)
The overall mean percentage of the spermatozoa with unstable chromatin was 4.6 ±
1.4 % (mean ± SD), with a minimum value of 3.1 % and a maximum value of 7.9 %.
About 70 % of the bulls (n = 21) recorded values less than 5 % and 30 % (n = 9)
showed higher values (> 5 %) of spermatozoa with unstable chromatin as shown in
figure 17.
4,13,93,4
4,54,1
3,1
3,6
3,4
4,34,5
3,23,73,4
5,25,4 4,6
5,4
6,4
7,6
4,4
3,23,3
4,1
5,9
6,1
3,5 3,34,1
7,2
7,8
-
1
2
3
4
5
6
7
8
9
10
11
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Bull No.
Spe
rmat
ozoa
with
uns
tabl
e ch
rom
atin
(%)
Figure 17: Percentage of spermatozoa with unstable Chromatin. (n = 90 ejaculates from 30 bulls, 3 ejaculates per bull).
4.2.3.1 Relationship between sperm chromatin status and IVF results
This experiment was carried to investigate the relationship between sperm chromatin
status and the results of in vitro fertilization (cleavage- and blastocyst-rate). Five
ejaculates from five different bulls were used. Two bulls (group I) had relatively low
mf-SCSA values (3.6 ± 0.7), and the other three bulls (group II) had relatively high
mf-SCSA values (7.6 ± 0.4) as shown in table 5. The selected bulls had apparently
normal classical spermatological parameters. Two bulls in the group 2 with a
relatively high abnormal sperm % and low alive sperm % were included in this
Results ___________________________________________________________________
69
experiment because there were no more bulls in this area (sperm with relatively high
instable chromatin). The standard spermatological parameters of the 5 bulls included
in this experiment are illustrated in table 5.
Item Bulls with low % unstable
sperm chromatin (group 1)
Bulls with high % unstable sperm
chromatin (group 2)
Bull No. 8 10 9 20 11
Chrom 4.1 3.1 3.6 ± 0.7 7.2 7.6 7.9 7.6 ± 0.4
Subj-f 75.0 66.7 70.9 ± 5.9 60 51.7 60 57.3 ± 4.8
Perc-f 88.4 81.7 85.1 ± 4.8 75 66.7 71.7 71.2 ± 4.2
Alive 80.9 66.0 73.5 ± 10.6 47.6 74.2 65.5 62.5 ± 13.5
Head 20.4 26.5 23.5 ± 4.4 32.4 37.2 23 30.9 ± 7.3
MAS 25.4 29.5 27.5 ± 2.9 39.4 48.9 33.5 40.6 ± 7.8
RVS 1.24 1.64 1.5 ± 0.3 1.5 1.7 1.44 1.6 ± 0.14
RVD -0.37 0.26 -0.05 ± 0.4 0.32 0.54 0.02 0.29 ± 0.26
VAP 66.0 66.5 66.3 ±0.4 68.6 61.0 63.1 64.3 ±4.0
VSL 57.7 58.4 58.1 ± 0.5 60.3 56.3 55.5 57.4 ± 2.6
Data are the mean value of three ejaculates per bull
Table 5: The sperm chromatin stability % and other spermatological parameters for two groups of bulls (values are averages of 3 ejaculates per bull (n = 5 bulls).
Percentage of spermatozoa with unstable chromatin Forward motile Spermatozoa (roughly estimated %) Forward motile percoll washed spermatozoa (roughly estimated %) Alive sperm (%) Sperm head abnormalities including acrosomal abnormalities (%) Morphologically altered spermatozoa (%) Modal value of relative volume shift Modal value of regulative volume decrease Average path velocity of spermatozoa (µm/sec.) Straight-line velocity of spermatozoa (µm/s)
Chrom Subj-f: Perc-f: Alive Head MAS RVS RVD VAP VSL
x x
Results ___________________________________________________________________
70
When the Pearson’s correlation coefficient estimated on the base of the mean values
of individual bulls, a significant correlation (p < 0.05) was obtained between mf-SCSA
value and cleavage rate, but not with blastocyst rate. Moreover comparison between
the two groups of bulls, revealed significance differences in blastocyst rate (P < 0.01)
and cleavage rate (P < 0.05) as shown in figure 18 and table 6.
Table 6: Cleavage- and blastocyst rates of individual bulls in relation to their chromatin instability % (n = 5 bulls). Group I: bulls with relatively low unstable sperm chromatin (3.6±0.7%). Group II: bulls with relatively high unstable sperm chromatin (7.6±0.4%).
Cleavage rate % Blastocyst rate%
Group Bull
No.
Chrom
Ooc
ytes
(n)
SD Min Max
SD Min Max
8 4.1 291 62.8 6.2 54.4 69.9 26.1 7.1 17.6 35.9
1
10 3.1 204 62.1 5.1 56.4 68.8 26.7 4.3 21.9 30.9
9 7.2 214 40.2 7.2 31.6 48.8 11.9 5.3 6.7 19.3
11 7.9 285 55.8 6.5 47.0 62.1 18.2 6.4 8.9 22.9 2
20 7.6 223 56.0 14.4 46.5 77.2 18.2 11.5 3.6 31.7
x x
Results ___________________________________________________________________
71
62.5a
26.4c
50.7b
16.0d
0
10
20
30
40
50
60
70
%
Group 1 Group 2
Cleav. R
Blast. R
Figure 18: Cleavage- and blastocyst rates in two groups of bulls. Group 1: bulls with relatively low instable sperm chromatin (3.6±0.7%). Group 2: bulls with relatively high instable sperm chromatin (7.6±0.4%). a, b : significantly different values (p < 0.05). c, d : significantly different values (p < 0.01).
4.3 OVIDUCTAL EXPLANT ASSAY (OEA)
Bull sperm attached rapidly to the oviductal explants. Despite gentle swirling after
sperm addition and before videotaping, attached spermatozoa were not evenly
distributed over the surfaces of the oviductal explants. They were spaced closely in
some areas, sparsely in others, and absent in a few areas. Sperm appeared to
adhere to the oviductal explants by rostral surface of the head and most of them
remained motile (98%). Viability of the oviductal explants was judged by vigorous
ciliary’s activity of ciliated cells. The ciliary’s beats were strong and were apparent
during the entire experiment. The overall mean of the binding indices was 15.1 ± 2.9
sperm / 0.01 mm2 (mean ± SD), with a range from 10.0 to 22.59.
Results ___________________________________________________________________
72
4.3.1 Differences among individual bulls
Statistical analysis of the data from 30 bulls (3 ejaculates per bull) revealed a
significant (P = 0.01) effect of individual bulls on the ability of sperm to bind to the
oviduct epithelial explants. Moreover significant (P � 0.05) differences were observed
among semen samples from different individual bulls in their binding indices. More
than 76 % of bulls (23 bulls) recorded binding indices between 12.2 and 18.0, while
only 10 % of bulls (3 bulls) recorded values more than 18.0 (mean + one SD), and
13.4 % of bulls (4 bulls) recorded values less than 12.2 (mean – one SD) as shown in
figure 19.
acde
b
abe
ace
ace
ab abd
abd
abd
abd abd
abd
abd ad acde
acde
acde ad
abd
cde cde
abd
abd
acde
c c
c
cde c
cde
-
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bull No.
Spe
rm (n
) / 0
,01m
m2 Exp
lant
(BI)
Figure 19: Sperm-oviduct explant Binding Indices of individual Bulls. (90 ejaculates from 30 bulls). Bulls with different letters have significant different binding indices.
Results ___________________________________________________________________
73
4.3.2 Relationship between sperm-oviduct binding ability and IVF
results
This experiment was carried out to investigate the relationship between sperm
oviduct binding ability and fertility in vitro. Six bulls were selected for this experiment,
three of them with relatively high BI and the others with relatively low BI. The IVF
parameters (cleavage and blastocyst rates) and BI of the selected bulls are listed in
table 7.
Due to the small number of the bulls used in this experiment, no correlations between
IVF results and BI were performed. However comparison between the 2 groups using
Wilcoxon two-sample test revealed a significant difference between the 2 groups in
blastocyst rate (P = 0.004), on the other hand, no significant (P = 0.48) difference
between the two groups in cleavage rate was recorded (figure 20). Concerning the
other spermatological parameters, no significant differences were recorded between
the 2 groups (table 8).
Table 7: Cleavage and blastocyst rates of two groups Bulls with low and high binding indices (n= 6 bulls).
Cleavage rate (%) Blastocyst rate (%)
Bul
l No
BI
Gro
up
Ooc
ytes
(n)
SD Min Max SD Min Max
1 19.3 293 61.5 16.9 40.7 80.4 10.0 3.8 6.3 15.2
3 17.9 312 63.7 10.7 47.7 75.9 18.4 2.9 15.9 23.7
15 22.5 1 (h
igh
BI)
309 53.3 3.1 51.0 58.5 21.1 4.3 17.7 26.5
25 10.0 330 53.5 13.3 34.7 69.7 9.5 4.7 3.9 15.2
26 10.2 346 70.1 6.9 60.6 78.6 12.0 5.8 5.4 19.0
27 10.9 2 (lo
w B
I)
309 64.5 11.6 47.9 79.7 11.0 2.7 8.4 15.2
x x
Results ___________________________________________________________________
74
Table 8: Sperm oviduct BI and spermatological parameters of two groups of bulls (n =
6). Values with different letters in the same row are significantly different. * = P � 0.05; ** = P � 0.001.
Item Group 1 (high BI) Group 2 (low BI)
Bull No. 1 3 15 X SD 25 26 27
X SD
BI 19.3 17.9 22.5 19.9a 2.4 10.0 10.2 10.9 10.4b 0.5
Chrom 3.4 4.0 4.5 4.0 0.6 4.1 5.9 6.2 5.4 1.2
Total 34.4 33.4 28.2 32.0 3.4 46.7 31.2 42.6 40.2 8.1
Head 30.0 22.5 25.4 26.0 3.8 38.4 27.5 38.9 35.0 6.5
Alive 69.6 73.5 72.8 72.0 2.1 66.8 64.6 54.8 62.1 6.4
RVS 1.9 1.8 2.2 1.97 0.21 1.7 1.2 1.4 1.5 0.25
RVD 0.51 0.69 0.85 0.68 0.17 0.50 -0.06 -0.1 0.11 0.34
Subj-f 63.4 68.4 71.7 67.9 4.2 50.0 60.0 55.0 55.0 5.0
Perc-f 81.7 80.0 88.4 83.4 4.5 65.0 65.0 60.0 63.4 2.9
VAP 67.2 63.2 54.2 61.6 6.7 59.6 65.8 62.6 62.8 3.0
VSL 57.0 55.9 55.6 56.2 0.8 52.8 58.3 57.4 56.2a 3.0
Sperm-oviduct binding index (sperm (n) / 0.01mm2 oviductal explant) Percentage of spermatozoa with unstable chromatin Morphologically altered spermatozoa (%) Sperm head abnormalities including acrosomal abnormalities (%) Alive sperm (%) Modal value of relative volume shift Modal value of regulative volume decrease Forward motile Spermatozoa (roughly estimated %) Forward motile percoll washed spermatozoa (roughly. Estimated %) Average path velocity of spermatozoa (µm/sec.) Straight-line velocity of spermatozoa (µm/s)
BI Chrom MAS Head Alive RVS RVD Subj-f Perc-f VAP VSL
Results ___________________________________________________________________
75
59.8
16.6a
62.7
10.9b
0
10
20
30
40
50
60
70
%
Group 1 Group 2
Cleav.RBlast.R
Figure 20: Cleavage and blastocyst rates in two groups of bulls with different sperm- oviduct explant binding indices (n = 6). Groupe 1: have high BI value (25.6). Groupe 2: have low BI value (8.3). a, b Significantly different values (p = 0.003).
4.4 CORRELATION MATRIX AMONG SPERMATOLOGICAL
PARAMETERS
Data of the 30 bulls included in this study were submitted to statistical analysis to
investigate the relationship between sperm oviduct interaction and both sperm cell
membrane functional integrity (m-HOST) and sperm chromatin status (mf-SCSA) as
well as other standard spermatological parameters. Pearson’s Correlation matrix of
all spermatological parameters is presented in table 9.
Results ___________________________________________________________________
76
4.4.1 Relationship between sperm-oviduct binding ability (BI) and
other spermatological parameters
1) Traditional spermatological parameters
No significant correlations were found between sperm oviduct binding capacity and
any of the conventional spermatological parameters. A highly significant correlation
(P = 0.0001) was recorded between BI and progressive forward motility of Percoll
washed spermatozoa.
2) Sperm membrane functional activity (m-HOST)
A significant positive correlation (P < 0.05) was recorded between binding ability of
sperm to oviduct epithelial explants and all sperm volumetric parameters calculated
from CASY1. The Pearson’s correlation coefficient was +0.50 for relative shift of
modal sperm cell volume (RVS) and +0.42 for regulative sperm cell volume decrease
(RVD).
3) Percentage of spermatozoa with unstable chromatin (mf-SCSA)
A significant (P = 0.02, r = -0.40) negative correlation was recorded between sperm
oviduct binding index and percentage of spermatozoa with unstable chromatin.
4.4.2 Relationship between sperm chromatin status (mf-SCSA) and other spermatological parameters
1) Conventional spermatological parameters
There was no significant correlation between percentage of spermatozoa with
unstable chromatin and any of conventional spermatological parameters. A
significant negative (P = 0.03) correlation was observed between mf-SCSA values
and forward motility % of percoll-selected spermatozoa.
2) Sperm membrane functional activity (m-HOST)
A significant negative correlation was observed between percentage of spermatozoa
with unstable chromatin and relative volume shift (RVS) of spermatozoa (p = 0.02).
Results ___________________________________________________________________
77
Meanwhile, no significant correlation was recorded between mf-SCSA value
(percentage of spermatozoa with unstable chromatin) and regulatory volume
decrease (RVD).
4.4.3 Relationship between sperm membrane functional status (m-
HOST) and standard spermatological parameters
There was no significant correlation between sperm volumetric parameters and any
of conventional spermatological parameters, except for progressive forward motility
% of Percoll washed spermatozoa, where a significant (P= 0.03) negative correlation
was detected.
Results ___________________________________________________________________
78
Item BI RVS RVD Subj-f Perc-f Alive Head MAS
r 0.50 RVS
p 0.004
r 0.42 0.78 RVD
p 0.02 .0001
r 0.20 0.19 0.04 Subj-f
p 0.30 0.33 0.83
r 0.70 0.40 0.19 Perc-f
p .0001 0.03 0.34
r 0.23 0.25 0.01 0.27 0.19 Alive
p 0.21 0.17 0.94 0.16 0.34
r -0.02 0.14 0.04 -0.77 -0.40 -0.03 Head
p 0.87 0.45 0.81 .0001 0.01 0.87
r -0.06 0.04 -0.01 -0.77 -0.45 -0.12 0.92 MAS
p 0.73 0.80 0.93 .0001 0.01 0.49 .0001
r -0.40 -0.41 -0.15 -0.12 -0.39 -0.16 -0.13 -0.01 chrom
p 0.02 0.02 0.40 0.50 0.03 0.37 0.48 0.94
Table 9: Pearson’s correlation matrix of both standard and advanced spermatological parameters.
Discussion ___________________________________________________________________
79
5 DISCUSSION
So far sperm oviduct interaction has been studied in many animal species using in
vitro coculture of spermatozoa with oviduct epithelial cells. These studies were mostly
focused on the mechanisms and biological aspects of sperm oviduct binding. The
present in vitro studies were conducted to further characterize the functional aspects
of the interaction between frozen-thawed bull spermatozoa and the oviductal
epithelium. Differences in the initial binding capacity of frozen-thawed sperm to
oviductal epithelium using the oviduct explant assay (OEA) were studied in repeated
ejaculates from 30 bulls. Additionally, the relationships between sperm-oviductal
epithelium binding capacity, membrane functional integrity and chromatin stability as
well as their relation to fertility in vitro were investigated.
5.1 OVIDUCT EXPLANT ASSAY (OEA)
Although several studies have shown that adhesion to the oviductal epithelium allows
the selection of spermatozoa characterized by intact acrosome (GUALTIERI and
TALEVI, 2000), an uncapacitated status (THOMAS et al., 1995a; LEFEBVRE and
SUAREZ 1996; FAZELI et al., 1999), low internal free calcium content and reduced
tyrosine phosphorylation of membrane proteins (DOBRINSKI et al., 1996;
PETRUNKINA et al., 2001a), superior morphology (THOMAS et al., 1994b) and
normal chromatin structure (ELLINGTON et al., 1999a), until now it is difficult to
determine whether these features of spermatozoa are associated with high
fertilization competence. It has been emphasized that for investigation of the sperm-
oviduct interaction, the in vitro conditions should mimic the in vivo situation as closely
as possible. Current methods for investigating the sperm–oviduct epithelial cell
interaction rely on the use of in vitro models, either oviductal epithelial explants (pigs:
SUAREZ et al., 1991; horse: THOMAS et al., 1994a; hamster: DE MOTT et al., 1995;
bovine: LEFEBVRE et al., 1995) or oviductal epithelial cells monolayers (bovine:
POLLARD et al., 1991 GUALTIERI and TALEVI, 2000; horse: DOBRINSKI et al.,
1996; THOMAS et al., 1995a; human: ELLINGTON et al., 1999a; porcine: FAZELI et
Discussion ___________________________________________________________________
80
al., 1999). Oviductal explants were used in this study, as it has been found that
explants mimic the in vivo situation and maintain most of the morphological
characteristics of oviductal epithelium (LEFEBVRE et al. 1995). Moreover the
oviductal explants were found to be physiologically the more responsive model than
oviduct epithelial cell monolayers (BUREAU et al., 2002).
In the current study the oviductal explants were obtained from both isthmus and
ampulla, since no significant regional effect of these two origins of explants could be
detected in vitro (pig: SUAREZ et al., 1991; PETRUNKINA et al., 2001b; cattle:
LEFEBVRE et al., 1995; hamster: SMITH and YANAGIMACHI, 1991).
The spermatozoa were co-incubated with oviductal epithelial explants for 15 min and
this incubation period resulted in detection of a representative number of
spermatozoa that attached to oviductal epithelial explants. GREEN et al. (2001)
reported that co-incubation periods < 15 min resulted in minimal binding of sperm to
oviductal epithelial cells, whereas only small increases in binding capacity were
detected after periods > 15 min.
We observed that spermatozoa attached to the epithelial surface of the explants
within minutes and most of them (> 95 %) remained motile, as they could be
identified by their long rapidly beating tails. Spermatozoa were found to bind mostly
by their heads and preferentially bind to the cilia of the oviductal cells and were not
evenly distributed over the surface. They were spaced closely in some areas, and
sparsely in others and were absent in a few areas, as has been observed previously
in dairy cattle (HUNTER et al., 1991; POLLARD et al., 1991), equine (THOMAS et
al., 1994a;b) and swine (RAYCHOUDHURY and SUAREZ, 1991; SUAREZ et al.,
1991). Our observations are inconsistent with THOMAS et al. (1994a) who reported
that stallion sperm seemed to bind equally as well to non-ciliated oviductal epithelial
cells and with HUNTER et al. (1991) who found that bull sperm attached to microvilli
of non-ciliated secretory oviductal cells in scanning electron micrographs of oviducts
of naturally mated cows. Accordingly, SMITH and YANAGIMACHI (1990) stated that
hamster spermatozoa were found to bind to both ciliated and secretory cells in the
oviductal epithelium.
Discussion ___________________________________________________________________
81
Differences among bulls in their capacity to establish a sperm reservoir probably exist
after mating or insemination, but it is difficult to detect these differences after
collection of oviducts because the number of sperm cells reported to reach the
oviduct in vivo has differed considerably among studies and within experiments
(SUAREZ et al., 1997; PARKER et al., 1975; MBURU et al., 1996). After using the
above discussed model assay (OEA), clear significant differences were detected
among individual semen samples in their initial binding capacity to the oviductal
epithelium, indicating the utility and suitability of this assay as a new semen
evaluation method. The individual differences of sperm binding capacity to the
oviductal epithelium has been reported also in vitro for stallion (THOMAS and BALL,
1996), boar (PETRUNKINA et al., 2001b) and bull (DE PAUW et al., 2002)
spermatozoa. These differences among bulls indicate a selective function of the
oviductal epithelium and the preferential initial binding of bull sperm to oviductal
epithelium in vitro might represent a mechanism for selecting functionally competent
sperm. The nature of the selective mechanism is not clear and may rely on the
differences in the expression of binding sites on the spermatozoa. At the time of
ejaculation, a coat of seminal plasma proteins becomes associated with the sperm
surface (TÖPFER-PETERSEN, 1999) and thus such proteins are likely to enter the
oviductal sperm reservoir where they could contribute to sperm–oviduct binding.
In the present study, the overall mean number of spermatozoa that bound to 0.01
mm2 explants was relatively lower than that reported in other studies (swine: GREEN
et al., 2001; cattle: DE PAUW et al., 2002). These differences may be either due to
different experimental conditions and/or to the method of data analysis.
5.1.1 Relationship between sperm-oviductal epithelium binding
capacity and chromatin stability
In the present study, a clear significant negative correlation was recorded between
the number of spermatozoa that bound to oviductal explants (BI) and the percentage
of spermatozoa with unstable chromatin in the ejaculate. This indicates that
spermatozoa with stable chromatin were preferentially selected by the oviductal
epithelium. These results support our previous findings (WABERSKI et al., 2003a),
Discussion ___________________________________________________________________
82
as the spermatozoa with stable chromatin in the Percoll selected sperm
subpopulation were found to preferentially bind to oviductal epithelium. Our results
are in agreement with those of ELLINGTON et al. (1999a, 2000) who observed a
selective binding of spermatozoa with stable chromatin to the oviductal epithelium in
human and bull semen after co-incubation with oviductal epithelial cell monolayers.
The selection mechanism in the female genital tract concerning the sperm chromatin
status seems to exist independently for sperm motility and sperm morphology,
because no correlations were detected between sperm chromatin status and
classical spermatological parameters.
It has been reported that co-culture of human sperm with bovine oviduct epithelial
cells reduces sperm chromatin structural changes that were observed during culture
in media alone (ELLINGTON et al., 1998a). The mechanisms by which the
spermatozoa with stable chromatin selectively bind to oviduct epithelial cells and the
prolongation of sperm chromatin stabilization when spermatozoa co-cultured with
oviductal epithelial cells are not fully understood. The maturation status of
spermatozoa was found to play an important role in the ability of spermatozoa to bind
to oviductal epithelium as it has been reported that epididymal spermatozoa and
spermatozoa with protoplasmic droplets (immature spermatozoa) were poorly bound
to oviductal epithelium compared with ejaculated (mature) boar spermatozoa
(PETRUNKINA et al., 2001b; MAGNUS, 2002; WABERSKI et al., 2003b).
Furthermore, the immature epididymal spermatozoa are characterized by not well
condensed (unstable) chromatin (BEDFORD et al., 1973; KOSOWER et al., 1992;
LÖHMER 2003). The results of the present study may indicate that a certain stage of
maturation is needed to complete the binding capacity of bull spermatozoa to
oviductal epithelium.
The freeze-thawing procedures can affect both sperm chromatin status and sperm
oviductal epithelium binding capacity, since it has been reported that
cryopreservation induces sub-lethal cellular damages in sperm including: partial
capacitation, loss of chromatin integrity, ultra-structural damage and accumulation of
reactive oxygen species (MAHADEVAN and TROUNSON, 1984; ALVAREZ and
STOREY, 1992; CORMIER et al., 1997; ELLINGTON et al., 1998a). Moreover
Discussion ___________________________________________________________________
83
ELLINGTON et al. (1999b) reported that human sperm attach to bovine OEC in vitro
in a dose-dependent manner. Additionally, binding capacity was decreased for
cryopreserved percoll-washed sperm although the motility did not differ (ELLINGTON
et al., 1999b). Furthermore, HAMMADEH et al. (1999) concluded that freeze-thawing
processes cause significant damage to chromatin condensation status, morphology
and membrane integrity of spermatozoa in both fertile and infertile men.
5.1.2 Relationship between sperm-oviductal epithelium binding
capacity and sperm volumetric parameters
To our knowledge this is the first trial to investigate the direct relationship between
the capacity of frozen-thawed bull spermatozoa to bind to oviductal epithelium (tested
in OEA) and sperm volume regulation ability (tested in m-HOST). Significant positive
correlations were obtained between the binding index (BI) and both RVS and RVD.
Sperm samples with high binding index responded well to hypo-osmotic stress (high
relative volume shift) and were characterised also by a high rate of regulatory volume
decrease when incubated for 20 min under hypo-osmotic conditions. These results
revealed that the functional integrity of sperm membrane plays a basic role in the
sperm-oviductal epithelium binding ability. In accordance, the results of UCHIDA et
al. (1992) revealed better in vivo ability of normal HOST human spermatozoa to
interact with the female reproductive tract.
This relationship could be discussed on the base of changes that occur during
cryostorage and capacitation processes, which may have an effect on both sperm
volume regulation ability and sperm-oviductal epithelium binding ability.
Changes in the plasma membrane after freeze–thawing procedures or cooling to low
temperatures have been reported to be similar to those that occur during capacitation
(WATSON, 1995; FULLER and WHITTINGHAM, 1997; GILLAN et al., 1997) and the
capacitation process was found to destabilize the sperm plasma membrane
(YANAGIMACHI, 1994; LANGLAIS and ROBERTS, 1985). Furthermore, during the
process of cryopreservation sperm are exposed to considerable changes in
osmolality, as well as mechanical cell injury could occur leading to intracellular or
extracellular ice crystal formation and signs of osmotic damage (WATSON, 1995;
Discussion ___________________________________________________________________
84
GAO et al., 1995; 1997). Furthermore it has been reported that freezing causes
extensive physical-chemical damage to the extracellular and intracellular membranes
of the sperm that are attributable to changes in the lipid phase transition and/or
increased lipid peroxidation (ALVAREZ and STOREY, 1992; 1993; MOSSAD et al.,
1994) during cooling or after thawing. HAMMADEH et al. (1999) reported that
cryoinjury to the plasma membrane in the tail region of human spermatozoa as
assessed by HOST was significantly higher after thawing in comparison to before
freezing. Moreover ELLINGTON et al. (1999b) reported that attachment of human
spermatozoa was decreased for cryopreserved sperm versus fresh sperm in a
manner separate from any differences in sperm motility. Furthermore DOBRINSKI et
al. (1995) reported that cryopreservation reduces the ability of equine spermatozoa to
attach to oviductal epithelial cell monolayers in vitro. Accordingly, more fresh sperm
were found to attach to OEC than that recorded for frozen-thawed semen in cattle
(GOLDMAN et al., 1998).
Concerning the sperm capacitation process and its effect on sperm volume regulation
and sperm oviduct binding, PETRUNKINA et al., (2004) reported that during
capacitation of boar sperm, a decrease in swelling level and disturbance of the
regulatory volume function were observed. These findings conform to those reported
previously (PETRUNKINA et al., 2000; PETRUNKINA and TÖPFER-PETERSEN
2000), where a decrease in relative volume swelling during incubation was observed
for both ejaculated and epididymal boar sperm exposed to capacitating conditions. It
has also been reported that uncapacitated bull (LEFEBVRE and SUAREZ, 1996) and
boar (FAZELI et al., 1999) spermatozoa were selectively and preferentially bound to
oviductal epithelium.
5.1.3 Relationship between sperm oviduct binding capacity and
fertility in vitro
In the present study, a clear significant difference was detected in the blastocyst rate
between the two selected groups of bulls; the first one was with relatively high
binding indices showed high blastocyst rate and the second one was with relatively
low binding indices showed low blastocyst rate. The individual bulls included in this
Discussion ___________________________________________________________________
85
experiment had normal standard spermatological parameters (Table 7). So the
significant difference in the blastocyst rate may rely only on the difference in binding
indices, not on the other sperm parameters.
The interpretation of this relationship could rely on the fact that spermatozoa, which
can bind to oviduct explants, are characterized by an uncapacitated status
(LEFEBVRE and SUAREZ, 1996), an intact acrosome (GUALTIERI and TALEVI,
2000), a superior morphology (THOMAS et al., 1994b) and a normal chromatin
structure (ELLINGTON et al., 1999a).
THUNDATHIL et al. (1999) demonstrated that the proportion of uncapacitated
spermatozoa present in frozen-thawed bull semen varies among bulls and more
important that the presence of uncapacitated spermatozoa is positively correlated
with fertility. Uncapacitated spermatozoa have an advantage compared to
capacitated spermatozoa during their transit to the site of fertilization in the oviduct,
because they are more likely to survive. If capacitation occurred before spermatozoa
reached the oviduct, the sperm population available for fertilization may be reduced
causing an adverse effect on fertilization. When the percentage of uncapacitated
spermatozoa in a sperm sample is high, more spermatozoa are able to bind to
oviduct explants, which may result in a higher fertility rate. Moreover, capacitation is
known to destabilize the sperm plasma membrane (YANAGIMACHI, 1994;
LANGLAIS and ROBERTS, 1985) and thus reduce the lifespan of spermatozoa
(WATSON, 1995). In a previous study which was carried out on cattle, DE PAUW et
al. (2002) demonstrated that the number of spermatozoa bound to oviduct epithelial
explants after 24 h of co-incubation was positively correlated with the non return rate
(NNR) when the membrane integrity of the initial sperm sample more than 60 %.
5.1.4 Relationship between sperm–oviduct binding ability and
standard spermatological parameters
The results of the present study revealed no significant correlation between the
binding index and any of the conventional spermatological parameters such as
motility, morphology and viability of spermatozoa. Although sperm motility is a well-
accepted criterion for ejaculate quality, the absence of correlation between motility
Discussion ___________________________________________________________________
86
and sperm oviduct binding capacity in the present study is not surprising. In vivo,
motility is important for overcoming the barriers in the female reproductive tract
(HUNTER, 1995; SUAREZ, 1996). However, sperm motility apparently has no crucial
importance for the quantitative success of sperm–oviduct binding in vitro, possibly
due to the free accessibility of explants. Furthermore in the present study the viable-
motile- and morphologically normal spermatozoa were selected by passing through
discontinuous percoll gradients. Our results are inconsistent with that reported by
PETRUNKINA et al. (2001b) who found a negative correlation between binding
indexes and both the percentage of morphologically abnormal spermatozoa and
percentage of spermatozoa with cytoplasmic droplets in boar semen. Our results also
disagreed with that reported by THOMAS et al. (1994b) who demonstrated that
spermatozoa of stallion ejaculates with low percentage of pathological alterations
bound to a higher extent to cultured epithelial cells than did spermatozoa of
ejaculates with a high percentage of such alterations. The species differences and
the type of semen preparation technique may account for these discrepancies. In the
present study frozen-thawed semen was used. Additionally high portions of dead and
morphologically abnormal spermatozoa were eliminated from the semen samples by
means of a discontinuous Percoll gradient centrifugation technique while in the
previous studies no Percoll-selected spermatozoa were used.
5.2 SPERM CHROMATIN STATUS (mf-SCSA)
Defective sperm DNA may not affect fertilization, but may cause great developmental
defects later. Consequently, it is important to employ sensitive procedures to select
bulls that produce sperm with high DNA integrity (SHEN et al., 1999; IRVINE et al.,
2000; VAN DER SCHANS et al., 2000).
The results of the present study revealed a narrower range (3.1 - 7.9 %) and a lower
overall mean percentage (4.6 ± 1.4 %) of spermatozoa with unstable chromatin in
semen samples than that recorded in many other studies.
It has been shown that the extent of bull sperm DNA denaturation in the samples
tested in SCSA varied from 5 % to 40 % (BALLACHEY et al., 1987) or even from 3 to
91.4 % (SAILER et al., 1996). Similar wide variations have been found in other
Discussion ___________________________________________________________________
87
studies on bull semen (EVENSON et al., 1980; BALLACHEY et al., 1988), also
BOCHENEK et al. (2001) reported that the proportion of bull spermatozoa with
defects in chromatin structure varied from 2.1 % to 23.8 %. Furthermore, EVENSON
et al. (1984) found that in human semen, spermatozoa with chromatin that was
susceptible to in situ denaturation can account for even 100 % and this is probably
associated with the fact that sperm chromatin is more sensitive to denaturation in
humans than in other species (LOVE and KENNEY, 1999). This may be due to the
fact that bulls in artificial insemination centres are generally selected for fertility,
therefore the percentage of spermatozoa with unstable chromatin are lower in bulls
than in other species like horse and human.
5.2.1 Relationship between sperm chromatin status (mf-SCSA) and
sperm volumetric parameters (m-HOST)
To our knowledge this is the first trial to investigate the direct relationship between
the sperm chromatin status estimated by mf-SCSA and the sperm volumetric
parameters (functional integrity of sperm membrane) estimated by a computer
assisted modified hypo-osmotic swelling test (m-HOST). A significant negative
correlation was found between relative volume shift (RVS) and percentage of
spermatozoa with unstable chromatin in the ejaculate. This relationship could be
discussed on the base of common factors, which may exert effects on both chromatin
condensation status and membrane integrity of spermatozoa in a given semen
sample, such as 1) the amount of reactive oxygen species (ROS) in the ejaculate, 2)
stress exerted on spermatozoa by freeze- thawing procedures and 3) the maturation
status of the spermatozoa in a given sample.
The generation of excess amounts of ROS by damaged human spermatozoa and
leukocytes has been implicated in the control of normal sperm function (AITKEN et
al., 1989a; DE LAMIRANDE and GAGNON, 1993) and in the aetiology of male
infertility associated with loss of plasma membrane function, altered motility, reduced
sperm–zona pellucida binding and acrosomal exocytosis (JONES et al., 1979;
ALVAREZ et al., 1987; AITKEN et al., 1989b, 1992). Additionally, it has been recently
reported that ROS (endogenously generated or provided as an exogenous stimulus)
Discussion ___________________________________________________________________
88
can cause an increase in DNA fragmentation in human spermatozoa (AITKEN et al.,
1989a; LOPES et al., 1998; TWIGG et al., 1998). Furthermore, the ROS have been
hypothesized to play a causative role in the aetiology of defective spermatozoa
function through peroxidation of the unsaturated fatty acids within the sperm plasma
membrane (AITKEN et al., 1992). Concerning the effect of freeze-thawing
procedures on both sperm membrane integrity and chromatin stability, it has been
reported that the cellular damages occurred during freezing are usually attributed to
membrane rupture caused by either the formation of intracellular ice crystals during
rapid cooling or by mechanical force from extra-cellular ice during slow cooling
(MOSSAD et al., 1994). It has previously been reported that freeze–thawing causes
significant damage to chromatin, membrane integrity and morphology of
spermatozoa in both fertile and infertile men (HAMMADEH et al., 1999, 2000). Other
researchers reported that freeze–thawing processes resulted in variations in the
compactness of mammalian spermatozoa nuclei and over-condensation of
spermatozoal DNA (ROYERE et al., 1988; ANZAR et al., 2002). These changes in
both sperm membrane and sperm DNA may account for the decreased conception
rates following insemination using frozen–thawed semen or for failure of conception
despite good post-thaw sperm motility. It has also been reported that loss of plasma
membrane asymmetry and occurrence of DNA fragmentation are early processes,
which precede the breakdown of plasma membrane integrity and loss of cell viability
during apoptosis (KOOPMAN et al., 1994; MARTIN et al., 1995; WYLLIE, 1980).
Moreover the presence of spermatozoa with damaged DNA in the ejaculate may be
indicative of the occurrence of apoptosis during spermatogenesis (BILLIG et al.,
1996; PENTIKAINEN et al., 1999; SAKKAS et al., 1999).
Concerning the maturation status of spermatozoa and its effect on both chromatin
status and membrane integrity, it has been emphasized that not only the nucleus but
also the sperm membrane are involved in the sperm maturation process in the
epididymis (AMANN et al., 1993; JONES, 1998). Furthermore, it has been reported
that the epididymal (immature) spermatozoa are characterized by not condensed-,
unstable chromatin (BEDFORD et al., 1973; LÖHMER, 2003).
Discussion ___________________________________________________________________
89
5.2.2 Relationship between sperm chromatin status and in vitro
fertility
The ability of sperm to fertilize is not only closely correlated to its morphology
(KRUGER et al., 1986) but also to the quality of the chromatin packaging (SAKKAS
et al., 1996; BIANCHI et al., 1996; MANICARDI, et al., 1995). Up till now the
available information about the possible fertility relevance of bull ejaculates with high
proportion of unstable sperm chromatin measured in SCSA relies on the non-return
rate (BALLACHEY et al. 1987; SAILER et al., 1996; BOCHENEK et al., 2001). In the
present experiment we used the IVF system to study the relationship between the
sperm chromatin status and fertility potential of five AI bulls because this system
offers the possibility to follow up the early in vitro embryo development up to the
blastocyst stage and postulate the disturbances in the embryo development that may
be caused by high percentage of spermatozoa with unstable chromatin. Although the
non return rate (NRR) in vivo fertility parameter have advantages, it is controversial
for the evaluation of the paternal fertility of a given bull, as it may be affected by many
factors such as age, parity and health status of the inseminated cows, inseminator,
as well as the insemination interval and breeding season. Moreover a very high
number of first inseminations will be needed to achieve it (AMANN and
HAMMERSTEDT, 2002). In addition, often no identical ejaculates are used for both
the in vitro semen analysis and the employment of insemination making the
relationship between spermatological investigation and the fertility more difficult
(AMANN and HAMMERSTEDT, 2002).
To ascertain the importance of the sperm chromatin stability for the fertility rate and
early embryo development, two groups of bulls, the first one (3 bulls) with extreme
high portion of spermatozoa with unstable chromatin and the second one (2 bulls)
with extreme low portion of spermatozoa with unstable chromatin were tested in an
IVF system. Sub-optimal sperm number (0.25 million sperm /ml) was used in order to
obtain clear differences in the fertilization capacity among bulls (AMANN and
HAMMERSTEDT, 2002). Significant differences were obtained in both cleavage
(day-3) and blastocyst (day-8) rates between the two tested bull groups, so a
Discussion ___________________________________________________________________
90
significant relationship between the IVF data and the mf-SCSA values could be
established. Our results are consistent with those of other studies (TEJADA et al.,
1984; IBRAHIM et al., 1988; BALLACHEY et al., 1987; GORCZYCA et al., 1993;
HOSHI et al., 1996; BOCHENEK et al., 2001), in which a significant relationship
between increased proportion of spermatozoa with unstable chromatin in the
ejaculate and lowered fertility was reported.
Although the fertilization rate (day-1) was not included in our study as in vitro fertility
parameters, the chromatin status seemed to induce adverse effect not only on the
cleavage and blastocyst rates but also on the fertilization and pregnancy rates. A
significant negative association was found between the percentage of sperm with
DNA fragmentation and fertilization rate (p = 0.008) and embryo cleavage rate (p =
0.01) after IVF (SUN et al., 1997), also a significant negative association was found
between the percentage of sperm with DNA fragmentation and the ICSI fertilization
rate (LOPES et al., 1998).
In contrast to the strong predictive value of the SCSA for negative pregnancy
outcome, fertilization rate in human was not found to be associated with sperm DNA
fragmentation or high DNA stainability (SAKKAS et al., 1996; TWIGG et al., 1998;
MORRIS et al., 2002; LARSON-COOK et al., 2003). This indicates that the chromatin
defect spermatozoa can penetrate the zona pellucida and fertilize the ova resulting in
loss of early embryos and negative pregnancy outcome (LARSON-COOK et al.,
2003).
5.3 SPERM VOLUMETRIC PARAMETERS (m-HOST)
The most important mechanisms of fertilization, such as capacitation, acrosome
reaction and binding of spermatozoa to the oocytes are believed to depend on the
functional integrity of the sperm membrane. Therefore, an appropriate assay set to
examine the fertilizing capacity of an ejaculate should include evaluation of the sperm
membrane integrity. The assessment of membrane integrity is based on examination
of the morphology, motility and hypo-osmotic swelling of spermatozoa (JEYENDRAN
et al., 1984), supravital stains and flowcytometric techniques (RODRIGUEZ-
MARTINEZ et al., 1997). In the current study significant differences among bulls in
Discussion ___________________________________________________________________
91
the two volumetric parameters (RVS and RVD) were detected. These results are
consistent with previous findings reported in bull by PETRUNKINA et al. (2001c) and
in human by PETZOLDT and ENGEL (1994) who mentioned that differences in cell
volume distribution among human ejaculates could be related to conventional sperm
parameters. In the present study we observed that the sperm volume remain
constant (stable) when spermatozoa were incubated in iso-osmotic HBS medium but
incubation of spermatozoa in hypo-osmotic HBS medium resulted in a quick swelling
of the sperm cells showing what is called relative volume shift (RVS). The sperm
volume reached to a maximum value at about 5 min, after that it was decreased
slowly in a curvilinear manner until reaching nearly to the original volume under iso-
osmotic conditions comprising what is called regulatory volume decrease (RVD).
These results are in agreement with the results reported previously in bull, boar and
dog semen (KULKARNI et al., 1997; PETRUNKINA et al., 2001c,d; PETRUNKINA et
al., 2004).
Although several different ion transport mechanisms may play a role in volume
regulation, in most mammalian cell types regulatory volume decrease is mediated
primarily via a volume-activated opening of independent K+ and Cl- channels.
KULKARNI et al. (1997), PETRUNKINA et al. (2001d) and YEUNG et al. (1999)
extend this deduction to bull, boar and mouse spermatozoa. As a cell swells in a
hypo-osmotic environment, the channels are activated to allow these ions to exit the
cell, thereby reducing internal osmolality and reversing swelling. Moreover our study
revealed a stability of the volumetric data on replicates of frozen-thawed ejaculates of
the same bull. The value of the relative volume shift provides a continuous
quantitative parameter of the osmotic response of individual bulls whereas the
classical HOST concerns only the percentage of a qualitative response (swollen or
non-swollen) to the hypo-osmotic conditions (PETRUNKINA et al., 2001d). Moreover,
the volume distribution shape was reported to be related to the spermatological
parameters of the human ejaculates (ENGEL and PETZOLDT, 1994 PETZOLDT and
ENGEL, 1994) and seems to be a crucial characteristic of semen quality both for
boar and bull semen as reported by LEIDING (1996).
Discussion ___________________________________________________________________
92
5.3.1 Relationship between sperm volumetric parameters (m-HOST) and viability of spermatozoa (SYBR14/PI)
The functional and structural integrity of sperm membrane are crucial for the viability
of spermatozoa. The commonly used staining test (eosin + nigrosin) for assessing
sperm membrane measures only its structural integrity. Recently a double, supravital
stain consists of PI and SYBR14 was used. The two stains, SYBR-14 (a green,
membrane-permeable stain) and PI (a red, membrane-impermeable counter stain)
have the same cellular target, the sperm DNA. The hypo-osmotic swelling test
(HOST) enables to evaluate the functional status of the sperm membrane. The
principle of HOST is based on water transport across the sperm tail membrane under
hypo-osmotic conditions.
The results of the present study revealed no correlation between the sperm volume
regulatory parameters RVS and RVD (functional integrity of sperm membrane) and
sperm viability assessed by SYBR14/PI (structural integrity of sperm membrane).
The lack of direct correspondence between the SYBR14/PI sperm viability assay and
sperm volume regulation ability indicate that both are independent assays, but both
assays indicate that sperm membrane damage is present and the number of
membrane-damaged spermatozoa varied among samples from different bulls. In
accordance, in several studies utilizing the HOST a sub-population of viable
spermatozoa with non-functional membrane was reported (VAZQUEZ et al., 1997;
PEREZ-LLANO et al., 2001; LECHNIAK et al., 2002). Besides HOST assess the
integrity of the plasma membrane along the sperm flagellum whereas fluorescent
dyes assess the head membrane. In contrast, the correlation between the
percentage of swollen sperm tails determined morphologically by the classical HOST
and the sperm viability determined by dual staining has been reported in canine
(RODRIGUEZ-GIL et al. 1994). In human VAN DER SAFFELE et al. (1992), reported
that the capacity of fresh spermatozoa to react in a hypo-osmotic environment
provided the same information as the viability test using eosin-nigrosin staining,
which evaluates the capacity of the head membrane to exclude dye. Furthermore, the
results of the present study revealed no significant correlations between sperm
volumetric parameters (RVS and RVD) and standard spermatological parameters
Discussion ___________________________________________________________________
93
(motility and morphology). In contrast to our results, it has been reported that sperm
volume distribution shape is related to classical spermatological parameters of
human ejaculates (VAN DER SAFFELE et al., 1992; ENGEL and PETZOLDT 1994;
PETZOLDT and ENGEL 1994). These discrepancies may be due to species
differences and/or experimental conditions.
In the present study, Percoll selected spermatozoa were used and it was established
that when semen sample passed through 2 layers of discontinuous Percoll gradients,
large portions of non-motile, morphologically altered and dead spermatozoa could be
eliminated (PARRISH et al., 1995).
5.3.2 Relationship between volumetric parameters (m-HOST) and
fertility (IVF)
Adequate numbers of progressively motile spermatozoa with biochemically active
membranes are required for successful fertilization (JEYENDRAN et al., 1984). In the
present study no correlation could be found between in vitro fertility parameters
(cleavage and blastocyst rates) and sperm volume regulatory parameters (RVS and
RVD). The results of the present study are consistent with that reported by CHAN et
al. (1988, 1989), BILJAN et al. (1996) and ROTA et al. (2000) who have found no
correlation between normal or abnormal classical HOST results and the success of in
vitro fertilization. On the contrary, our results are inconsistent with that reported in bull
by PETRUNKINA et al. (2001c) who used the NRR as fertility parameter and in
human by (CHECK et al., 1995; VED et al., 1997; ZEYNELOGLU et al., 2000;
TARTAGNI et al., 2002).
VAN DER VEN et al., (1986) found a close correlation between the percentage of
swollen human spermatozoa and the percentage of denuded hamster oocytes that
were penetrated by capacitated spermatozoa. In bovine the suitability of the HOST
as a predictive tool for in vitro fertility has not yet been established. Furthermore the
HOST evaluates the membrane function, but other variables may be implicated in the
fertility potential, especially under in vitro conditions. This may explain the absence of
a significant correlation between m-HOST parameters and in vitro fertility parameters
in the present study. The type of fertilization procedure, data interpretation, and
Discussion ___________________________________________________________________
94
statistical analysis may account for these discrepancies. Recently, it has been
hypothesized that a defect in the functional integrity of the sperm membrane, which is
detectable by the HOST, may reduce fertility potential by causing implantation
disorders rather than fertilization problems (CHECK et al., 2001). Subtle
abnormalities in sperm detected with the HOST may lead to subsequent abnormal
membrane function in the embryo and anomalies in the cell-to-cell communication
and binding that seem to have an important role in the attachment of the blastocyst
and subsequent penetration of the surface epithelium of the endometrium (DENKER,
1993).
5.4 CONCLUSION
The individual bull difference in their capacity to bind to oviductal epithelium indicates
a selective function of sperm-oviduct binding. It is also suggested that an increased
percentage of spermatozoa with unstable chromatin coincides with a disturbed
plasma membrane functions as indicated by altered sperm volume regulation and
reduced BI. Moreover the determination of the capacity of sperm to bind to oviductal
epithelium could become a reliable in vitro method for predicting the fertility of a given
sire. Furthermore, it can also be concluded that the modified fluorescence
microscopical sperm chromatin structure assay is a reliable test to detect sperm
chromatin stability as an independent fertility relevant parameter in the bull.
Summary ___________________________________________________________________
95
6 SUMMARY
Abdel-Tawab Abdel-Razek Yassin Khalil
BINDING CAPACITY OF BULL SPERMATOZOA TO OVIDUCTAL EPITHELIUM IN
VITRO AND ITS RELATION TO SPERM CHROMATIN STABILITY, SPERM
VOLUME REGULATION AND FERTILITY
The present study was undertaken to evaluate the efficiency of the sperm-oviduct
explant binding assay (OEA) in predicting the sperm fertilization potential in vitro and,
to investigate the relationship between the sperm oviduct binding ability, sperm
response to hypo-osmotic stress and sperm chromatin stability.
90 ejaculates from 30 test bulls were submitted to: the Oviductal-Explant-Assay
(OEA), modified fluorescence microscopical Sperm-Chromatin Structure Assay (mf-
SCSA), modified Hypo-Osmotic Swelling Test (m-HOST) and viability assessment
using SYB14/Probidium Iodide double staining (Sperm Viability Kits). As well as were
submitted to morphological and motility evaluation using Cell Motion Analyser (CMA).
The number of spermatozoa that bound to 0.01 mm2 of oviductal explant (Binding
index; BI) was used as the parameter of binding capacity.
Significant (p<0.05) differences in BI and sperm volumetric parameters among bulls
were reported in the present study. The BI was positively significant correlated with
all sperm volumetric parameters (RVS and RVD) that recovered from the m-HOST
and negatively significant (p<0.05) correlated with percentage of spermatozoa with
unstable chromatin. No significant correlations were found between the BI and most
of the conventional spermatological parameters. The percentage of spermatozoa with
unstable chromatin was significantly (P<0.05) negative correlated with relative
volume increase, but not correlated with regulative volume decrease (RVD). No
significant correlations were recorded between most of the conventional
spermatological parameters and both sperm volumetric parameters and percentage
Summary ___________________________________________________________________
96
of spermatozoa with unstable chromatin, except for forward motility % of Percoll
selected spermatozoa, where a significant (p<0.05) negative correlation was
recorded. Six ejaculates of bulls with low (n = 3) and high (n = 3) binding index and
five ejaculates of bulls with low (n = 2) and high (n = 3) percentage of spermatozoa
with unstable chromatin were further examined for their fertility by the in vitro
fertilization (IVF) test. Concerning the relation between the BI and fertility in vitro, no
significant difference in cleavage rate was recorded between the two groups of bulls,
while a significant (p<0.01) difference in blastocyst rate was recorded, as the bulls
with high BI (26.6±5.8) showed high blastocyst rate (16.6±5.8) and the bulls with low
BI (8.3±1.7) showed low blastocyst rate (10.9±1.3).
Regarding the relationship between chromatin stability and in vitro fertility, significant
differences in both cleavage rate (P<0.05) and blastocyst rate (P<0.01) were
recorded between the two groups of bulls, where the bulls with low percentages (2.8
±0.5) of sperm with unstable chromatin showed significantly higher cleavage- and
blastocyst rate (62.5±0.5, 26.4±5.7 respectively) than bulls with high percentages of
sperm with unstable chromatin (9±0.8) that showed cleavage rate of 50.7±9.1 and
blastocyst rate of 16.1±7.8.
In conclusion, the individual bull difference in the BI indicates a selective function of
sperm oviduct binding. It is also suggested that an increased percentage of
spermatozoa with unstable chromatin coincides with a disturbed plasma membrane
function as indicated by altered sperm volume regulation and reduced BI. Moreover
determination of the capacity of sperm to bind oviductal epithelium could become a
reliable in vitro method for predicting the fertility of a given sire.
Zusammenfassung
___________________________________________________________________
97
7 ZUSAMMENFASSUNG
Abdel-Tawab Abdel-Razek Yassin Khalil
BINDUNGSFÄHIGKEIT VON BULLENSPERMIEN AN OVIDUKTEPITHEL IN
VITRO UND DEREN BEZIEHUNG ZUR SPERMIENCHROMATINSTABILITÄT,
VOLUMENREGULIERUNG UND FRUCHTBARKEIT IN VITRO
Ziel der vorliegenden Studie war, eine mögliche bullenspezifische Bindungsfähigkeit
von Spermien an Oviduktepithel in vitro zu untersuchen und das Verhältnis zwischen
der Bindungsfähigkeit der Samenzellen und der Fähigkeit zur Volumenregulation
unter hypoosmotischen Bedingungen sowie zur Stabilität des Spermienchromatins zu
ermitteln.
Tiefgefrorenes Sperma von 90 Ejakulaten von 30 Testbullen wurden hinsichtlich der
Bindungsfähigkeit im Ovidukt Explant Assay (OEA), der Widerstandsfähigkeit der
Spermien DNA gegenüber saurer Denaturierung in situ mit dem modifizierten
fluoreszenzmikroskopischen Spermien-Chromatin-Struktur-Assay (mf-SCSA) und der
volumenregulatorischen Spermienmembranfunktion mit dem modifizierten Hypo-
Osmotischen Schwell-Test (m-HOST) untersucht. Zusätzlich wurde die
Spermienmorphologie, die computergestützte Motilität sowie die Permeabilität der
Plasmammembran für die Farbstoffe SYBR14/Propidium Jodid (®Sperm viability Kits)
ermittelt. Mit Sperma ausgewählter Bullen wurde ein in vitro-Fertilisationstest
durchgeführt.
Es gab eine signifikante (p<0,05) positive Korrelation zwischen dem Bindungsindex
und volumetrischen Parametern (RVSund RVD). Zwischen dem Bindungsindex und
dem Prozentsatz an Spermien mit instabilem Chromatin bestand eine signifikante
(p<0,05) negative Korrelation. Es gab eine signifikante (P<0,05) negative Korrelation
zwischen den Prozentsatz von Spermien mit instabilem Chromatin und der relativen
Volumenverschiebung (RVS). Dahingegen konnte keine signifikante Korrelation
Zusammenfassung
___________________________________________________________________
98
zwischen der Chromatinstabilität und der regulativen Volumenabnahme (RVD)
festgestellt werden
Bindungsindex, Volumenregulationsparameter sowie Chromatinstabilität zeigten
keine Korrelation zu den standardspermatologischen Parametern.
Sechs Ejakulate von sechs Bullen mit niedrigem (n = 3) und hohem (n = 3)
Bindungsindex (BI) und fünf Ejakulate von fünf Bullen mit niedrigem (n = 2) und
hohem (n = 3) Prozentsatz an Spermien mit instabilem Chromatin wurden hinsichtlich
ihrer Fertilität im in vitro Befruchtung Test (IVF) überprüft. Bullen mit hohem
Bindungsindex (26.6±5.8) wiesen signifikant (p<0,01) höhere Blastozystenraten auf,
als Bullen mit niedrigem BI (8.3±1.7); zwischen den Teilungsraten bestand kein
signifikanter Unterschied. Bullen mit niedrigen Prozentsätzen chromatininstabiler
Spermien (2.8±0.5 %) zeigten signifikant (p<0,01) höhere Teilungs- und
Blastozystenraten als Bullen mit erhöhtem Vorkommen chromatininstabiler Spermien
(9±0.8 %).
Folgende Schlussfolgerungen werden gezogen: Mit dem Ovidukt Explant Assay steht
möglicherweise ein Testsystem zu Verfügung, welches bullenspezifische,
fertilitätsrelevante Eigenschaften an der Plasmamembran von Spermien erfasst, die
durch standardspermatologische Parameter nicht diagnostizierbar sind. Ein erhöhter
Prozentsatz von Spermien mit instabilem Chromatin geht mit einer gestörten
Plasmamembranfunktion einher, wie durch die veränderte
Spermienvolumenregulation im hypoosmotischen Schwelltest und verringerten
Bindungsindex im Ovidukt Explant Assay angezeigt wird.
References
___________________________________________________________________
99
8 REFERENCES ABE H. and T. OIKAWA (1993): Observations by scanning electron microscopy of oviductal epithelial cells from cows at follicular and luteal phases. Anat. Rec. 235, 399-410 ABE H., M. ONODERA and S. SUGAWARA (1993): Scanning electron microscopy of goat oviductal epithelial cells at the follicular and luteal phases of the oestrus cycle. J. Anat. 183, 415-421 ABE H., Y. SENDAI, T. SATOH and H. HOSHI (1995a): Bovine oviduct-specific glycoprotein: a potent factor for maintenance of viability and motility of bovine spermatozoa in vitro. Mol. Reprod. Dev. 42, 226-232 ABE H., Y. SENDAI, T. SATOH and H. HOSHI (1995b): Secretory products of bovine oviductal epithelial cells support the viability and motility of bovine spermatozoa in culture in vitro. J. Exp. Zool. 272, 54-61 ABU-MUSA A., K. TAKAHASHI and M. KITAO (1993): Correlation between postswim-up hypoosmotic swelling test and in vitro fertilization results. Int. J. Fertil. 38, 113 –116 ACEVEDO, N., J.H. BAME, L.E. KUEHN, W.D. HOHENBOKEN, D.P. EVENSON and R.G. SAACKE (2001): Effects of scrotal insulation on morphology and chromatin characteristics of ejaculated spermatozoa in the bovine. In: Proceedings of the Conference of Society for the Study of Reproduction, Ottawa, Canada, July 29 . Aug 1, 2001 ADAMS C.E. and M.C. CHANG (1962): Capacitation of rabbit spermatozoa in the Fallopian tube and in the uterus. J. Exp. Zool. 15, 159-166 AHLGREN M. (1975): Sperm transport to and survival in the human fallopian tube. Gynecol. Invest. 6, 206-214, Review AHUJA K.K. (1985): Carbohydrate determinants involved in mammalian fertilization. Am. J. Anat. 174, 207-223
References
___________________________________________________________________
100
AITKEN R.J. and H. FISHER (1994): Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bio. Essays, 16, 259–267 AITKEN R.J., D. BUCKINGHAM, K. WEST, F.C. WU, K. ZIKOPOULOS and D.W. RICHARDSON (1992): Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J. Reprod. Fertil. 94, 451–462 AITKEN R.J. and J.S. CLARKSON (1987): Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil. 81, 459–469 AITKEN R.J., J.S. CLARKSON and S. FISHEL (1989a): Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41, 183–197 AITKEN R.J., J.S. CLARKSON, T.B. HARGREAVE, D.S. IRVINE and F.C. WU (1989b): Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J. Androl. 10, 214-220 ALVAREZ J.G. and B.T. STOREY (1992): Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a model of sublethal cryodamage to human sperm during cryopreservation. J. Androl. 13, 232-241 ALVAREZ J.G. and B.T. STOREY (1993): Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm—glycerol and other polyols as role cryoprotectant. J. Androl. 14,199–209. ALVAREZ J.G., R.K. SHARMA, M.OLLERO, R.A. SALEH, M.C. LOPEZ, A.J. THOMAS JR, D.P. EVENSON and A. AGARWAL (2002): Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay. Fertil. Steril. 78, 319-329
References
___________________________________________________________________
101
ALVAREZ J.G., J.C.TOUCHSTONE, L. BLASCO and B.T. STOREY (1987): Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa; Superoxide dismutase as a major enzyme protectant against oxygen toxicity. J. Androl. 8, 338–348 AMANN R.P. (1989): Can the fertility potential of a seminal sample be predicted accurately? J. Androl. 10, 89-98 AMANN R.P. and R.H. HAMMERSTEDT (1993): In vitro evaluation of sperm quality: an opinion. J Androl. 14, 397-406, Review AMANN R.P. and R.H. HAMMERSTEDT (2002): Detection of differences in fertility. J. Androl. 23, 317-325, Review AMANN R.P., R.H. HAMMERSTEDT and D.N. VEERAMACHANENI (1993): The epididymis and sperm maturation: a perspective. Reprod. Fertil. Dev. 5, 361-381, Review AMSO N.N., J. CROW, J. LEWIN and R.W. SHAW (1994): A comparative morphological and ultrastructural study of endometrial gland and fallopian tube epithelia at different stages of the menstrual cycle and the menopause. Hum Reprod. 9, 2234-22341 ANDERSON S.H. and G.J. KILLIAN (1994): Effect of macromolecules from oviductal conditioned medium on bovine sperm motion and capacitation. Biol Reprod. 51, 795-799 ANGELOPOULOS T., Y.A.MOSHEL, L.LU, E. MACANAS, J.A. GRIFO and L.C. KREY (1998): Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization. Fertil. Steril. 69, 740-747 ANZAR M., L. HE, M.M. BUHR, T.G. KROETSCH and K.P. PAULS (2002): Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility. Biol Reprod. 66, 354-360
References
___________________________________________________________________
102
ARAVINDAN G.R., BJORDAHL J., JOST L.K. and EVENSON D.P. (1997): Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp Cell Res. 236, 231-237 ARMSTRONG J.S., M. RAJASEKARAN, W. CHAMULITRAT, P. GATTI, W.J. HELLSTORM and S.C. SIKKA (1999): Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radical Biol. Med. 26, 869–880 ARVER S. and R. ELIASSON (1982): Zinc and zinc ligands in human seminal plasma. II. Contribution by ligands of different origin to the zinc binding properties of human seminal plasma. Acta Physiol Scand. 115, 217-224 ASCHKENAZI J., R. ORVIETO, R.GOLD-DEUTSCH, D.FELDBERG, D.DICKER, I.VOLIOVITCH and Z. BEN-RAFAEL (1992): The impact of woman’s age and sperm parameters on fertilization rates in IVF cycles. Eur. J. Obstet. Gynecol. Reprod. Biol. 66, 155-159 AUGER J., M. MESBAH, C. HUBER and J.P. DADOUNE (1990): Aniline BLUE staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men. Int. J. Androl. 13, 452-462 AVERY S., U.M. BOLTON and B.A. MASON (1990): An evaluation of the hypoosmotic sperm swelling test as a predictor of fertilizing capacity in vitro. Int. J. Androl. 13, 93–99 AYAD V.J., S.A. MCGOFF and D.C. WATHES (1990): Oxytocin receptors in the oviduct during the oestrous cycle of the ewe. J. Endocrinol. 124, 353-359 BADER H. (1982): An investigation of sperm migration into the oviducts of the mare. J. Reprod. Fertil. Suppl. 32, 59-64 BAILLIE H.S., A.A. PACEY, M.A.WARREN, I.W. SCUDAMORE and C.L. BARRATT (1997): Greater numbers of human spermatozoa associate with endosalpingeal cells derived from the isthmus compared with those from the ampulla. Hum Reprod. 12, 1985-1992
References
___________________________________________________________________
103
BAKST M.R. (1994): Fate of fluorescent stained sperm following insemination: new light on oviductal sperm transport and storage in the turkey. Biol. Reprod. 50, 987-992 BALHORN R. (1982): A model for the structure of chromatin in mammalian sperm. J. Cell. Biol. 93, 298-305 BALHORN R., M. CORZETT, J. MAZRIMAS and B. WATKINS (1991): Identification of bull protamine disulfides. Biochemistry. 30, 175-181 BALLACHEY B.E., D.P. EVENSON and R.G. SAACKE (1988): The sperm chromatin structure assay: Relationship with alternate tests of semen quality and heterospermic performance of bulls. J Androl. 9, 109-115 BALLACHEY B.E., W.D. HOHENBOKEN and D.P. EVENSON (1987): Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility. Biol Reprod. 36, 915-925 BALLACHEY B.E., W.D. HOHENBOKEN and D.P. EVENSON (1986): Sperm head morphology and nuclear chromatin structure evaluated by flow cytometry in a diallel cross in mice. Can. J. Genet. Cytol. 28, 954-966 BALL B.A., I. DOBRINSKI, M.S. FAGNAN and P.G. THOMAS (1997): Distribution of glycoconjugates in the uterine tube (oviduct) of horses. Am. J. Vet. Res. 58, 816-822 BALL B.A. (1996): Scanning electron microscopy of the equine oviduct and observations on ciliary currents in vitro at DAY 2 after ovulation. Theriogenology 46, 1305-1311 BARRATT C.L.R. and I.D. COOKE (1991) Sperm transport in the human female reproductive tract--a dynamic interaction Int. J. Androl. 14, 394-411, Review BARRATT C.L.R., J.C. OSBORN, P.E. HARRISON, N.MONLESS, B.C.DUMPHY, E.A. LENTON and I.D. COOKE (1989): The hypoosmotic swelling test and the sperm mucus penetration test in determining fertilization of the human oocyte. Hum Reprod. 4, 430–434
References
___________________________________________________________________
104
BASTIAS M.C., H. KAMIJO and K.G. OSTEEN (1993): Assessment of human sperm functional Changes after in-vitro coincubation with cells retrieved from the human female reproductive tract. Hum. Reprod. 8, 1670 -1677 BATTUT I., J. BEZARD and E. PALMER (1991): Establishment of equine oviduct cell monolayers for co-culture with early equine embryos. J. Reprod. Fertil. Suppl. 44, 393-403 BECKMAN, K.B. and B.N. AMES (1997): Oxidative decay of DNA. J..Biol..Chem. 272, 19633-19636 BEDFORD J.M. and W.G. BREED (1994): Regulated storage and subsequent transformation of spermatozoa in the fallopian tubes of an Australian marsupial, Sminthopsis crassicaudata. Biol. Reprod. 50, 845-854 BEDFORD J.M. (1983): Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol Reprod. 28, 108-20, Review BEDFORD J.M. and H.I. CALVIN (1974): Changes in -S-S- linked structures of the sperm tail during epididymal maturation, with comparative observations in sub-mammalian species. J. Exp. Zool. 187, 181-204 BEDFORD J.M., M.J. BENT and H. CALVIN (1973): Variations in the structural character and stability of the nuclear chromatin in morphologically normal human spermatozoa. J. Reprod. Fertil. 33, 19-29 BELETTI M.E. and M.L.S. MELLO (1996): Methodological variants contributing to detection of abnormal DNA-protein complexes in bull spermatozoa . Brazilian Journal of Genetics 19, 97-103 BELLVÈ A.R., R. CHANDRIKA and A.D. BARTH (1990): Temporal expression, polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis. J. Cell Sci. 96, 745-756
References
___________________________________________________________________
105
BENAVENTE R. and G. KROHNE (1985): Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line. Proc. Natl. Acad. Sci. U S A. 82, 6176-6180 BENNETT W.A., T.L. WATTS, W.D. MLAIR, S.J. WALDHALM and J.W. FUQUAY (1988): Patterns of oviducal motility in the cow during the oestrous cycle. J. Reprod. Fertil. 83, 537-543 BIANCHI P.G., G.C. MANICARDI, F. URNER, A. CAMPANA and D. SAKKAS (1996): Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa. Mol Hum Reprod. 2, 139-144 BILJAN M.M., W.M. BUCKETT, C.T. TAYLOR, M.LUCKAS, I. AIRD, C.R. KINGSLAND and D.I. LEWIS-JONES (1996): Effect of abnormal hypo-osmotic swelling test on fertilization rate and pregnancy outcome in in vitro fertilization cycles. Fertil. Steril. 66, 412-416 BILLIG H., S.Y. CHUN, K. EISENHAUER and A.J. HSUEH (1996): Gonadal cell apoptosis: hormone-regulated cell demise. Hum. Reprod. Update. 2, 103-117,Review BJORNDAHL L. and U. KVIST (1990): Influence of seminal vesicular fluid on the zinc content of human sperm chromatin. Int. J. Androl. 13, 232-237 BLANDAU R.J. and P. GADDUM-ROSSE (1974): Mechanism of sperm transport in pig oviducts. Fertil. Steril. 25, 61-67 BLAZAK W.F. and J.W. OVERSTREET (1982): Instability of nuclear chromatin in ejaculated spermatozoa of fertile men. J. Reprod. Fertil. 65, 331-339 BLEIL J.D. and P.M. WASSERMAN (1983): Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. Biol. 95, 317-324 BLEIL J.D. and P.M. WASSERMAN (1980): Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20, 873-882
References
___________________________________________________________________
106
BLOTTNER S., C. WARNKE, A. TUCHSCHERER, V. HEINEN and H. TORNER (2001): Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season. Anim. Reprod. Sci. 65, 75-88 BOATMAN D.E. and G.E. MAGNONI (1995): Identification of a sperm penetration factor in the oviduct of the golden hamster. Biol. Reprod. 52, 199-207 BOCHENEK M., Z. SMORAG and J. PILCH (2001): Sperm chromatin structure assay of bulls qualified for artificial insemination. Theriogenology 56, 557-567 BOILARD M., J.BAILEY, S. COLLIN, M. DUFOUR and M.A. SIRARD (2002): Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach. Biol. Reprod. 67, 1125-1132 BOLLO E., B. BIOLATTI, S. PAU and M. GALLONI (1990): Scanning electron microscopy of pathologic Changes in the epithelial surfaces of the uterus and uterine tubes of cows. Am. J. Vet. Res. 51, 137-42 BONGSO A., HO J., C.Y. FONG, S.C. NG and S. RATNAM (1993): Human sperm function after coculture with human fallopian tubal epithelial cell monolayers: in vitro model for studying cell interactions in early human conception. Arch. Androl. 31, 183-190 BOYLE M.S., D.G.CRAN, W.R. ALLEN and R.H. HUNTER (1987): Distribution of spermatozoa in the mare's oviduct. J. Reprod. Fertil. Suppl. 35, 79-86 BRENNER R.M., J.A. RESKO and N.B. WEST (1974): Cyclic Changes in oviductal morphology and residual cytoplasmic estradiol binding capacity induced by sequential estradiol--progesterone treatment of spayed Rhesus monkeys. Endocrinology 95, 1094-104 BREWER L.R., M. CORZETT and R. BALHORN (1999): Protamine-induced condensation and decondensation of the same DNA molecule. Science 286, 120–123 BRINSKO S.P., D.D. VARNER, T.L. MEYERS and S.A. MEYERS (1990): The effect of postbreeding uterine lavage on pregnancy rate in mares Theriogenology 33, 465-475
References
___________________________________________________________________
107
BRINSKO S.P., D.D. VARNER and T.L. MEYERS (1991): The effect of uterine lavage performed four hours postinsemination on pregnancy rates in mares. Theriogenology 35, 1111-1119 BUCKETT W.M. (2003): Predictive value of hypo-osmotic swelling test to identify viable non-motile sperm Asian. J. Androl. 5, 209-212 BUREAU M., J.L.BAILEY and M. A. SIRARD (2002): Binding Regulation of Porcine Spermatozoa to Oviductal Vesicles In vitro. J. Androl. 23, 188–193 CALVIN H.I. and J.M. BEDFORD (1971): Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Fertil. Suppl. 13, 65-75 CASEY P.J., R.B. HILLMAN, K.R. ROBERTSONB, A.I.YUDIN, I.K.M. LIU and E.Z. DROBNIS (1993): Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm. J. Androl. 14, 289–297 CHAN P.J., D.R. TREDWAY, J. CORSELLI, S.C. PANG and B.C. SU (1991): Combined supravital staining and hypoosmotic swelling test. Hum. Reprod. 6, 1115-1118 CHAN S Y W., C. WANG, S.T.H. CHAN and P.C. HO (1990): Differential evaluation of human sperm hypoosmotic swelling test and its relationship with the outcome of in-vitro fertilization of human oocytes. Hum Reprod. 5, 84–88 CHAN S.Y.W., C. WANG, M. NG, G. TAM, T. LO, W.L.TSOI, G. NIE and J. LEUNG (1989): Evaluation of computerized analysis of sperm movement characteristics and differential sperm tail swelling patterns in predicting human sperm in vitro fertilizing capacity. J. Androl. 10, 133-138 CHAN S.Y.W., C. WANG, M. NG, W.W. SO and P.C. HO (1988): Multivariate discriminant analysis of the relationship between the hypo-osmotic swelling test and the in-vitro fertilizing capacity of human sperm. Int. J. Androl. 11, 369-378
References
___________________________________________________________________
108
CHAN S.Y.W., E.J. FOX, M.M.C. CHAN, W.L. TSOI, C. WANG, L.C.H.TANG, G.W.K. TANG. and P.C. HO (1985): The relationship between the human sperm hypo-osmotic swelling test, routine semen analysis, and the human sperm zona-free hamster ovum penetration assay. Fertil. Steril. 44, 668–672 CHECK J.H., A. BOLLENDORF, M. PRESS and T. BLUE (1992): Standard sperm morphology as a predictor of male fertility potential. Arch. Androl. 28, 39-41 CHECK J.H., L. STUMPO, D. LURIE, K. BENFER and C. CALLAN (1995): A comparative prospective study using matched samples to determine the influence of subnormal hypo-osmotic test scores of spermatozoa on subsequent fertilization and pregnancy rates following in-vitro fertilization. Hum. Reprod. 10, 1197-1200 CHECK J.H., D. KATSOFF and M.L. CHECK (2001): Some semen abnormalities may cause infertility by impairing implantation rather than fertilization. Med. Hypotheses 56, 653-657 CHIAN R.C. and M.A. SIRARD (1995): Fertilizing ability of bovine spermatozoa cocultured with oviduct epithelial cells. Biol. Reprod. 52, 156-162 CHIAN R.C., S. LAPOINTE and M.A. SIRARD (1995): Capacitation in vitro of bovine spermatozoa by oviduct epithelial cell monolayer conditioned medium. Mol. Reprod. Dev. 42, 318-324 CLAASSENS O.E., R. MENKVELD, D.R. FRANKEN, E.PRETORIUS, Y. SWART, C.J. LOMBARD and T.F. KRUGER (1992): The Acridine Orange test: determining the relationship between sperm morphology and fertilization in vitro. Hum. Reprod. 7, 242-247 CODDINGTON C.C., S.C. OEHNINGER, D.L. OLIVE, D.R. FRANKEN, T.F. KRUGER and G.D. HODGEN (1994): Hemizona index (HZI) demonstrates excellent predictability when evaluating sperm fertilizing capacity in in vitro fertilization patients. J. Androl. 15, 250-254 COETZEE K., T.F. KRUGER, R. MENKVELD, C.J. LOMBARD and R.J. SWANSON (1989): Hypo-osmotic swelling test in the prediction of male fertility. Arch. Androl. 23, 131–138
References
___________________________________________________________________
109
COOPER T.G. and C.H. YEUNG (1998) A flow cytometric technique using peanut agglutinin for evaluating acrosomal loss from human spermatozoa. J. Androl. 19, 542–550 CORDOVA A., J.F. PEREZ-GULTIERRE, B. LLEO, C. GARCIA-ARTIGA, A. ALVAREZ, V. DROBCHAK and S. MARTIN-RILLO (2002): In vitro fertilizing capacity and chromatin condensation of deep frozen boar semen packaged in 0.5 and 5 ml straws. Theriogenology 57, 2119-2128 CORMIER N., M. A. SIRARD and J.L., BAILEY (1997): Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J. Androl. 18, 461-468 CORREA J.R., M.M. PACE and P.M. ZAVOS (1997): Relationships among frozen-thawed sperm characteristics assessed via routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology 48, 721–731 CREW F.A.E. (1922): A suggestion as to the cause of the aspermatic condition of the imperfectly descended testis. J..Anat. 56, 98 CUMMING I.R. (1995): Suitability of the intact acrosome method for the prediction of fertility in bovine artificial insemination. Vet. Rec. 136, 289-291 CUMMINS J.M., A.M. JEQUIER and R. KAN (1994): Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress. Mol. Reprod. Dev. 37, 345–362 CUMMINS J.M. and R. YANAGIMACHI (1982): Sperm-egg ratios and the site of the acrosome reaction during in vitro fertilization in hamster. Gamete Res. 5, 239–256 D'AGATA R., E. VICARI, M.L. MONCADA, G. SIDOTI, A.E. CALOGERO, M.C. FRONITO, G. MINACAPILLI, A. MONGIOI and P. POLOSA (1990): Generation of reactive oxygen species in subgroups of infertile men. Int. J. Androl. 13, 344–351
References
___________________________________________________________________
110
DARZYNKIEWICZ Z., G. JUAN, X.LI, W. GORCZYCA, T. MURAKAMI and F. TRAGANOS (1997): Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1-20, Review DARZYNKIEWICZ Z., F. TRAGANOS, T. SHARPLESS and M.R. MELAMED (1975): Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp. Cell Res. 90, 411-428 DEBARLE M., A. MARTINAGE, P. SAUTIERE and P. CHEVAILLIER (1995): Persistence of protamine precursors in mature sperm nuclei of the mouse. Mol. Reprod. Dev. 40, 84-90 DE JONGE C.J., C.L. BARRATT, E. RADWANSKA and I.D. COOKE (1993): The acrosome reaction-inducing effect of human follicular and oviductal fluid. J. Androl. 14, 359–365 DE LAMIRANDE E. and C. GAGNON (1993): Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma. Fertil. Steril. 59, 1291-1295 DE MOTT R.P. and S.S. SUAREZ (1992): Hyperactivated sperm progress in the mouse oviduct. Biol. Reprod. 46, 779-785 DE MOTT R.P., R. LEFEBVRE and S.S. SUAREZ (1995): Carbohydrates mediate the adherence of hamster sperm to oviductal epithelium. Biol. Reprod. 52, 1395-1403 DENKER H.W. (1993): Implantation: a cell biological paradox. J. Exp. Zool. 266, 541–558 DE PAUW I.M., A. VAN SOOM, H. LAEVENS, S. VERBERCKMOES and A. DE-KRUIF (2002): Sperm binding to epithelial oviduct explants in bulls with different nonreturn rates investigated with a new in vitro model. Biol. Reprod. 67, 1073-1079 DICKENS C.J., J. SOUTHGATE and H.J. LEESE (1993): Use of primary cultures of rabbit oviduct epithelial cells to study the ionic basis of tubal fluid formation. J. Reprod. Fertil. 98, 603-610
References
___________________________________________________________________
111
DOBRINSKI I., H.P. HUGHES and A.D. BARTH (1994): Flow cytometric and microscopic evaluation and effect on fertility of abnormal chromatin condensation in bovine sperm nuclei. J. Reprod. Fertil. 101, 531-538 DOBRINSKI I., T.T. SMITH, S.S. SUAREZ and B.A. BALL (1997): Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biol. Reprod. 56, 861-869 DOBRINSKI I., S.S. SUAREZ and B.A. BALL (1996): Intracellular calcium concentration in equine spermatozoa attached to oviductal epithelial cells in vitro. Biol. Reprod. 54, 783-788 DOBRINSKI I., P.G. THOMAS and B.A. BALL (1995): Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro. J. Androl. 16, 536-542 DREVIUS L.O. (1972): Bull spermatozoa as osmometers. J. Reprod. Fertil. 28, 29-39 DREVIUS L.O. and H. ERIKSSON (1966): Osmotic swelling of mammalian spermatozoa. Exp. Cell Res. 42, 136-56 DUBUC A. and M.A. SIRARD (1995): Effect of coculturing spermatozoa with oviductal cells on the incidence of polyspermy in pig in vitro fertilization. Mol. Reprod. Dev. 41, 360-367 DU J., J. TAO, F.W. KLEINHAUS, A.T. PETER and J.K. CRISTER (1994): Determination of boar spermatozoa water volume and osmotic response. Theriogenology 42, 1183–1191 DURAN E.H., T. GURGAN, S. GUNALP, M.E. ENGINSU, H. YARALI, and A. AYHAN (1998): A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum. Reprod. 13, 1235-1239 EDWARDS L.J. and H.J. LEESE (1993): Glucose transport and metabolism in rabbit oviduct epithelial cells. J. Reprod. Fertil. 99, 585-591
References
___________________________________________________________________
112
EGGERT-KRUSE W., G. ROHR, H. KERBEL, B. SCHWALBACH, T. DEMIRAKCA, K. KLINGA, W. TILGEN and B. RUNNEBAUM (1996a): The Acridine Orange test: a clinically relevant screening method for sperm quality during infertility investigation. Hum. Reprod. 11, 784-789 EGGERT-KRUSE W., H. SCHWARZ, G. ROHR, T. DEMIRAKCA, W. TILGEN and B. RUNNEBAUM (1996b): Sperm morphology assessment using strict criteria and male fertility under in-vivo conditions of conception. Hum. Reprod. 11, 139-146 EHRENWALD E., R.H. FOOTE and J.E. PARKS (1990): Bovine oviductal fluid components and their potential role in sperm cholesterol efflux. Mol. Reprod. Dev. 25, 195-204 ELLINGTON J.E., B.A. BALL, B.J. BLUE and C.E. WILKER (1993a): Capacitation-like membrane Changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Am. J. Vet. Res. 1505-1510 ELLINGTON J.E., B.A. BALL and X. YANG (1993c): Binding of stallion spermatozoa to the equine zona pellucida after coculture with oviductal epithelial cells. J. Reprod. Fertil. 98, 203-208 ELLINGTON, J.E., D.P. EVENSON, J. DE AVILA, L.K. JOST and R.W. WRIGHT (2000): Bull sperm that attach to oviduct cells in vitro support superior embryonic develop -ment rates as compared to sperm that do not attach. In: Proceedings of the 14th International Congress of Animal Reproduction, Stockholm, Vol.1, 2 - 6. July ELLINGTON J.E., L. D. BROEMELING, S.J. BRODER, A.E. JONES, D. A. CHOKER and R.W. WRIGHT. (1999b): Comparison of fresh and cryopreserved human sperm attachment to bovine oviduct (uterine tube) epithelial cells in vitro. J. Androl. 20, 492-949 ELLINGTON J.E., D.P. EVENSON, J.E. FLEMING, R.S. BRISBOIS, G.A. HISS, S.J. BRODER and R.W. WRIGHT JR (1998a): Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural Changes seen during culture in media alone. Fertil. Steril. 69, 643-649
References
___________________________________________________________________
113
ELLINGTON J.E., D.P. EVENSON, R.W. WRIGHT JR, A.E. JONES, C.S. SCHNEIDER, G.A. HISS and R.S. BRISBOIS (1999a): Higher-quality human sperm in a sample selectively attach to oviduct (fallopian tube) epithelial cells in vitro. Fertil. Steril. 71, 924-929 ELLINGTON J.E., G.G. IGNOTZ, B.A. BALL, V.N. MEYERS-WALLEN and W.B. CURRIE (1993d): DE novo protein synthesis by bovine uterine tube (oviduct) epithelial cells Changes during co-culture with bull spermatozoa. Biol. Reprod. 48, 851-856 ELLINGTON J.E., G.G. IGNOTZ, D.D. VARNER, R.S. MARCUCIO, P. MATHISON and B.A. BALL (1993b): In vitro interaction between oviduct epithelial and equine sperm. Arch. Androl. 31, 79-86 ELLINGTON J.E., A.E. JONES, C.M. DAVITT, C.S. SCHNEIDER, R.S. BRISBOIS, G.A. HISS and R.W. JR-WRIGHT (1998b): Human sperm function in co-culture with human, macaque or bovine oviduct epithelial cell monolayers. Hum. Reprod. 13, 2797-2804 ELLINGTON J.E., V.N. MEYERS-WALLEN and B.A. BALL (1995): Establishment of a coculture system for canine sperm and uterine tube epithelial cells. Vet. Rec. 27, 542-543 ELLINGTON J.E., A.W. PADILLA, W.L. VREDENBURGH, E.P. DOUGHERTY and R.H. FOOTE (1991): Behaviour of bull spermatozoa in bovine uterine tube epithelial cell co-culture: an in vitro model for studying the cell interactions of reproduction. Theriogenology 36, 977–989 ENGEL S. and R. PETZOLDT (1994): The spermatozoal volume as indicative of the plasma membrane integrity (modification of the hypoosmotic swelling test) 1 Methods. Andrologia 26, 309 313 ENGINSU M.E., J.C.M. DUMOULIN, M.H.E.C. PIETERS, M. BERGERS, J.L.H. EVERS and J.P.M. GERAEDTS (1992): Comparison between the hypoosmotic swelling test and morphology evaluation using strict criteria in predicting in vitro fertilization (IVF). J. Assist. Reprod. Genet. 9, 259–264
References
___________________________________________________________________
114
ENGLE C.E., C.W. FOLEY, D.M. WITHERSPOON, R.D. SCARTH and D.D. GOETSCH (1975): Influence of mare uterine tubal fluids on the metabolism of stallion sperm. Am. J. Vet. Res. 36, 1149-1152 ESTERHUIZEN A.D., D.R. FRANKEN, J.G. LOURENS, E. PRINSLOO and L.H. VAN ROOYEN (2000a): Sperm chromatin packaging as an indicator of in-vitro fertilization rates. Hum. Reprod. 15, 657-661 ESTERHUIZEN A.D., D.R. FRANKEN, J.G. LOURENS, C. VAN ZYL, I.I. MULLER and L.H. VAN ROOYEN (2000b): Chromatin packaging as an indicator of human sperm dysfunction. J. Assist. Reprod. Genet. 17, 508-514 ESTEVES S.C., R.K. SHARMA, A.J. THOMAS-JR and A. AGARWAL (1996): Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm. Fertil. Steril. 66, 798-804 ESTOP A.M., S. MUNNE, L.K. JOST and D.P. EVENSON (1993): Studies on sperm chromatin structure alterations and cytogenetic damage of mouse sperm following in vitro incubation. Studies on in vitro-incubated mouse sperm. J. Androl. 14, 282-288 EVENSON D.P. (1999a): Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects. Reprod. Fertil. Dev. 11, 1-15, Review EVENSON D.P. (1999b): Alternations and damage of sperm chromatin structure and early embryonic failure, in: Towards Reproductive Certainty: Fertility and Genetics Beyond 1999. JANNSEN R, MORTIMER D (eds) Parthenon Publishing Group Ltd, New York, pp 313–329 EVENSON D.P. (1997): Sperm nuclear DNA strandbreaks and altered chromatin structure: Are there concerns for natural fertility and assisted fertility in the andrology lab? Moving Beyond Boundaries: Clinical Andrology in the 21st Century. Andrology, Laboratory, Workshop. Postgraduate Course. Baltimore, MD. EVENSON D.P., Z. DRAZYNKIEWICZ and M.R. MELAMED (1980): Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210, 1131–1133
References
___________________________________________________________________
115
EVENSON D.P., P.J. HIGGINS, D. GRUENEBERG and B.E. BALLACHEY (1985): Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry 6, 238-253 EVENSON D.P. and L.K. JOST (2000): Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci. 22, 169-189 EVENSON D P. and L.K. JOST (1994): Sperm chromatin structure assay: DNA denaturability. Methods Cell Biol. 42 Pt B: 159-176 EVENSON D.P., L.K. JOST, R.K. BAER, T.W. TURNER and S.M. SCHRADER (1991): Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod. Toxicol. 5, 115-125 EVENSON D.P., L.K. JOST, M. CORZETT and R. BALHORN (2000): Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study. J. Androl. 21, 739-746 EVENSON D.P., L.K. JOST, D. MARSHALL, M.J. ZINAMAN, E. CLEGG, K. PURVIS, P. DE-ANGELIS and O.P. CLAUSSEN (1999): Utility of the sperm chromatin structure assay (SCSA) as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod. 14, 1039–1049 EVENSON D.P., F.A. KLEIN, W.F. WHITMORE and M.R. MELAMED (1984): Flow cytometric evaluation of sperm from patients with testicular carcinoma. J. Urol. 132, 1220-1225 EVENSON D.P., K.L. LARSON and L.K. JOST (2002): Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J. Androl. 23, 25-43, Review EVENSON D.P. and M.R. MELAMED (1983): Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J. Histochem. Cytochem. 31(1A Suppl), 248-253
References
___________________________________________________________________
116
EVENSON D.P., L. THOMPSON and L.K. JOST (1994): Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology 41, 637–651 EVREV T.I., A.I. VAWDA and V.B. MAKAVEEVA (1994): Effect of tubular and follicular fluids on acrosin activity and sperm motility. Arch. Androl. 32, 207-212 FAWCETT D.W., W.A. ANDERSON and D.M. PHILLIPS (1971): Morphogenetic factors influencing the shape of the sperm head. Dev. Biol. 26, 220-225 FAZELI A., A.E. DUNCAN, P.F. WATSON and W.V. HOLT (1999): Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species. Biol. Reprod. 60, 879-886 FETTEROLF P.M. and B.J. ROGERS (1990): Prediction of human sperm penetrating ability using computerized motion parameters. Mol. Reprod. Dev. 27, 326-331 FLECHON J.E. and R.H. HUNTER (1981): Distribution of spermatozoa in the utero-tubal junction and isthmus of pigs, and their relationship with the luminal epithelium after mating: a scanning electron microscope study. Tissue Cell 13, 127-139 FOSSA S.D., P.ANGELIS, S.M.KRAGGERUD, D.P. EVENSON, L. THEODORSEN and O.P. CLAUSEN (1997): Prediction of posttreatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay. Cytometry 30, 192-196 FRASER L.R., L.R. ABEYDEERA and K. NIWA (1995): Ca2+-regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis. Mol. Reprod. Develop. 40, 233–241 FULLER S.J. and D.G. WHITTINGHAM (1997): Capacitation-like changes occur in mouse spermatozoa cooled to low temperatures. Mol. Reprod. Dev. 46,318-324. FUSE H., T. KAZAMA and T. KATAYAMA (1991): Relationship between hypoosmotic swelling test, semen analysis, and zona-free hamster ovum test. Arch. Androl. 27, 73–78
References
___________________________________________________________________
117
GADDUM-ROSSE P. and R.J. BLANDAU (1976): Comparative observations on ciliary currents in mammalian oviducts. Biol. Reprod. 14, 605-609 GAGNON C., A. IWASAKI, E. DE-LAMIRANDE and N. KOVALSKI (1991): Reactive oxygen species and human spermatozoa. Ann. NY Acad. Sci. 637, 436–444 GAHMBERG C.G., P. KOTOVUORI and E. TONTTI (1992): Cell surface carbohydrate in cell adhesion. Sperm cells and leukocytes bind to their target cells through specific oligosaccharide ligands. APMIS Suppl. 27, 39-52 Review GAO D.Y., C. LIU, L.E. MC-GANN, P.F.WATSON, F.W.KLEINHAUS, P. MAZUR, E.S. CRITSER and J.K. CRITSER (1995): Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod 10, 1109–1122 GAO D.Y., P. MAZUR and J.K. CRITSER (1997): Fundamental cryobiology of mammalian spermatozoa. In Karow AM, Critser JK (eds), Reproductive Tissue Banking. Academic Press, London, pp. 263–328. GARNER D.L. and L.A. JOHNSON (1995): Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod. 53, 276-284 GARNER D.L., L.A. JOHNSON, S.T. YUE, B.L. ROTH and R.P. HAUGLAND (1994): Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J. Androl. 15, 620-629 GARNER D.L., D. PINKEL, L.A. JOHNSON and M.M. PACE (1986): Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Biol. Reprod. 34, 127-138 GILLAN L., G. EVANS and W.M. MAXWELL (1997): Capacitation status and fertility of fresh and frozen-thawed ram spermatozoa. Reprod. Fertil. Dev. 9,481-487 GILMORE J.A., J. DU, J. TAO, A.T. PETER and J.K. CRISTER (1996): Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J. Reprod. Fertil. 107, 87-95
References
___________________________________________________________________
118
GOGOL P., M. BOCHENEK and Z. SMORAG (2002): Effect of rabbit age on sperm chromatin structure. Reprod. Domest. Anim. 37, 92-95 GOLDMAN E.E., J.E. ELLINGTON and R.H. FOOTE (1998): Reaction of fresh and frozen bull spermatozoa incubated with fresh and frozen bovine oviduct epithelial cells. Reprod. Nutr. Dev. 38, 281-828 GORCZYCA W., F. TRAGANOES, H. JESIONOWSKA and Z. DRAZYNKIEWICZ (1993): Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp. Cell. Res. 207, 202-205 GREEN C.E., J. BREDL, W.V. HOLT, P.F. WATSON and A. FAZELI (2001): Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro. Reproduction 122, 305-315 GREEN G.R., R. BALHORN, D.L. POCCIA and N.B. HECHT (1994): Synthesis and processing of mammalian protamines and transition proteins. Mol. Reprod. Dev. 37, 255-263 GRIPPO A.A., S.H. ANDERSON, D.A. CHAPMAN, M.A HENAULT and G.J. KILLIAN (1994): Cholesterol, phospholipid and phospholipase activity of ampullary and isthmic fluid from the bovine oviduct. J. Reprod. Fertil. 102, 87-93 GRIPPO A.A., A.L. WAY and G.J. KILLIAN (1995): Effect of bovine ampullary and isthmic oviductal fluid on motility, acrosome reaction and fertility of bull spermatozoa. J. Reprod. Fertil. 105, 57-64 GROW D.R., S.OEHNINGER, H.J SELTMAN, J.P. TONER, R.J. SWANSON, T.F. KRUGER and S.J. MAUSHER (1994): Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a large in vitro fertilization population. Fertil. Steril. 62, 559-567 GUALTIERI R. and R. TALEVI. (2000): In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release. Biol. Reprod. 62, 1754-1762
References
___________________________________________________________________
119
GUALTIERI R. and R. TALEVI (2003): Selection of highly fertilization-competent bovine spermatozoa through adhesion to the Fallopian tube epithelium in vitro. Reproduction 125, 251-258 GUERIN J.F., N. OUHIBI, G. REGNIER-VIGOUROUX and Y. MENEZO (1991): Movement characteristics and hyperactivation of human sperm on different epithelial cell monolayers. Int. J. Androl. 14, 412-422 GUNN R.M.C., R.N. SAUNDERS and W. GRANGER (1942): Seminal changes affecting fertility in sheep. Bull Counc. Sci. Industr. Res. Aust. 148 GUTIERREZ A., J. GARDE, C. GARCIA-ARTIGA and I. VAZQUEZ (1993): Ram spermatozoa cocultered with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction. Mol. Reprod. Dev. 36, 338-345 GUYADER C. and D. CHUPIN (1991): Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cell monolayers. Theriogenology 36, 505-512 HACKETT A.J. and J.W. MACPHERSON (1965): A method for differential staining of bovine spermatozoa after extension in sterile milk. Can. Vet. J. 111, 117-120 HAMMADEH M.E., A.S. ASKARI, T. GEORG, P. ROSENBAUM and W. SCHMIDT (1999): Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int. J. Androl. 22, 155-162 HAMMADEH M.E., C.DEHN, M. HIPPACH, T. ZEGINIADOU, M.STIEBER, T. GEORG, P. ROSENBAUM and W. SCHMIDT. (2001): Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men. Int. J. Androl. 24, 66-72 HAMMADEH M.E., D.C. NKEMAYIM, T. GEORG, P. ROSENBAUM and W. SCHMIDT. (2000): Sperm morphology and chromatin condensation before and after semen processing. Arch. Androl. 44, 221-226
References
___________________________________________________________________
120
HAMAMAH S., D. ROYERE, J.C. NICOLLE, M. PAQUIGNON and J. LANSAC (1990): Effects of freezing thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species. Reprod. Nutr. Dev. 30, 59-64 HAMMERSTEDT R.H., A.D. KEITH, S. HAY, N. DELUCA and R.P. AMANN (1979): Changes in ram sperm membranes during epididymal transit. Arch. Biochem. Biophys. 196, 7-12 HANCOCK J.L. (1951): A staining technique for the study of temperature shock in semen. Nature 167, 323 HARKEMA W., R.A.P. HARRISON, N.G.A. MILLER, E.K. TOPPER and H. WOELDERS (1998): Enhanced binding of zona pellucida proteins to the acrosomal region of intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study. Biol. Reprod. 58, 421–430 HARPER M.J. (1994): Gamete and zygote transport. In: The physiology of Reproduction. 2nd Ed, Knobil E and Neil JD (Ed), Raven Press, Ltd., New York, 123-187 HARRISON R.A. and S.E. VICKERS (1990): Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88, 343-352 HARRISON R.A.P., B. MAIRET and N.G.A. MILLER (1993): Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations. Mol. Reprod. Dev. 35, 197-208 HAUSER R., H. YAVETZ, G.F. PAZ, Z.T. HOMONNAI, A. AMIT, J.B.LESSING, M.R. PEYSER and L. YOGEV (1992): The predictive fertilization value of the hypo-osmotic swelling test (HOST) for fresh and cryopreserved sperm. J. Assist. Reprod. Genet. 9, 265–270 HAWK H.W. (1987): Transport and fate of spermatozoa after insemination of cattle. J. Dairy Sci. 70, 1487-1503, Review HENAULT M.A. and G.J. KILLIAN (1993): Synthesis and secretion of lipids by bovine oviduct mucosal explants. J. Reprod. Fertil. 98, 431-438
References
___________________________________________________________________
121
HESS H., H. HEID and W.W. FRANKE (1993): Molecular characterization of mammalian cylicin, a basic protein of the sperm head cytoskeleton. J. Cell. Biol. 122, 1043-1052 HOSHI K., H. KATAYOSE, K. YANAGIDA, Y. KIMURA and A. SATO (1996): The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil. Steril. 66, 634-639 HUD N.V., M.J. ALLEN, K.H. DOWNING, J. LEE and R. BALHORN (1993): Identification of the elemental packing unit of DNA in mammalian sperm cells by atomic force microscopy. Biochem. Biophys. Res. Commun. 193, 1347–1354 HUGHES C.M., S.E. LEWIS, V.J. MCKELVEY-MARTIN and W. TOMPSON (1998): The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum. Reprod. 13, 1240–1247 HUGHES C.M., V.J. MCKELVEY-MARTIN and S.E. LEWIS (1999): Human sperm DNA integrity assessed by the Comet and ELISA assays Mutagenesis. 14, 71-75 HUNTER R.H. (1973): Polyspermic fertilization in pigs after tubal deposition of excessive numbers of spermatozoa. J. Exp. Zool. 183, 57-63 HUNTER R.H. (1981): Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation. J. Reprod. Fertil. 63, 109-17 HUNTER R.H. (1984): Pre-ovulatory arrest and peri-ovulatory redistribution of competent spermatozoa in the isthmus of the pig oviduct. J. Reprod. Fertil. 72, 203-211 HUNTER R.H. (1991): Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. Mol. Reprod. Dev. 29, 385-91, Review HUNTER R.H. (1995): Human sperm reservoirs and Fallopian tube function: a role for the intra-mural portion. Acta Obstet. Gynecol. Scand. 74, 677-681, Review
References
___________________________________________________________________
122
HUNTER R.H. (1998) Have the Fallopian tubes a vital role in promoting fertility? Acta Obstet. Gynecol. Scand. 77, 475-486, Review HUNTER R.H., B. FLECHON and J.E. FLECHON (1991): Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study. Tissue Cell 23, 641-656 HUNTER R.H., L. BARWISE and R. KING (1982): Sperm transport, storage and release in the sheep oviduct in relation to the time of ovulation. Br. Vet. J. 138, 225-232 HUNTER R.H., B. FLECHON and J.E. FLECHON (1987): Pre- and peri-ovulatory distribution of viable spermatozoa in the pig oviduct: a scanning electron microscope study. Tissue Cell 19, 423-436 HUNTER R.H. and J.P. HALL (1974): Capacitation of boar spermatozoa: synergism between uterine and tubal environments. J. Exp. Zool. 188, 203-213 HUNTER R.H. and P.C. LEGLISE (1971): Polyspermic fertilization following tubal surgery in pigs, with particular reference to the role of the isthmus. J. Reprod. Fertil. 24, 233-246 HUNTER R.H. and R. NICHOL (1983): Transport of spermatozoa in the sheep oviduct: preovulatory sequestering of cells in the caudal isthmus. J. Exp. Zool. 228, 121-128 HUNTER R.H. and R. NICHOL (1988): Capacitation potential of the fallopian tube: a study involving surgical insemination and the subsequent incidence of polyspermy. Gamete Res. 21, 255-266 HUNTER R.H., R. NICHOL and S.M. CRABTREE (1980): Transport of spermatozoa in the ewe: timing of the establishment of a functional population in the oviduct. Reprod. Nutr. Dev. 1980; 20, 1869-1875
References
___________________________________________________________________
123
HUNTER R.H. and I. WILMUT (1984): Sperm transport in the cow: peri-ovulatory redistribution of viable cells within the oviduct. Reprod. Nutr. Dev. 24, 597-608 IBRAHIM M.E., M.A. MOUSSA and H. PEDERSEN (1988): Sperm chromatin heterogeneity as an infertility factor. Arch. Androl. 1988; 21:129–133 IBRAHIM M.E. and H. PEDERSEN (1988): Acridine orange fluorescence as male fertility test. Arch. Androl. 20, 125-129 ICHIMURA S., M. ZAMA and H. FUJITA (1971): Quantitative determination of single-stranded sections in DNA using the fluorescent probe acridine orange. Biochim. Biophys. Acta. 240, 485-495 IJAZ A., R.D. LAMBERT and M.A. SIRARD (1994): In vitro-cultured bovine granulosa and oviductal cells secrete sperm motility-maintaining factor (s). Mol. Reprod. Dev. 37, 54-60 IRANPOUR F.G., M.H. NASR-ESFAHANI, M.R. VALOJERDI and T.M. AL.-TARAIHI (2000): Chromomycin A3 staining as a useful tool for evaluation of male fertility. J. Assist. Reprod. Genet. 17, 60-66 IRVINE D.S., I.C. MACLEOD, A.A. TEMPLETON, A. MASTERTON and A. TAYLOR (1994): A prospective clinical study of the relationship between the computer-assisted assessment of human semen quality and the achievement of pregnancy in vivo. Hum. Reprod. 9, 2324-2334 IRVINE D.S., J.P. TWIGG, E.L. GORDON, N. FULTON, P.A. MILNE and R.J. AITKEN (2000): DNA integrity in human spermatozoa: relationships with semen quality. J. Androl. 21, 33-44 ITO M., T.T. SMITH and R. YANAGIMACHI (1991): Effect of ovulation on sperm transport in the hamster oviduct. J. Reprod. Fertil. 93, 157-163 JANSEN R.P. (1984): Endocrine response in the fallopian tube. Endocr. Rev. 5, 525-551, Review
References
___________________________________________________________________
124
JANUSKAUSKAS A., A. JOHANNISSON, L. SODERQUIST and H. RODRIGUEZ-MARTINEZ (2000): Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls. Theriogenology 53, 859–875 JEYENDRAN R.S., W.J. HOLMGREN, P. BIELFELD and A.C. WENTZ (1992): Fertilizing capacity of various populations of spermatozoa within an ejaculate. J. Assist. Reprod. Genet. 9, 32-35 JEYENDRAN R.S., H.H. VAN DER VEN, M. PEREZ-PELAEZ, B.G. CRABO and L.J. ZANEVELD (1984): Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil. 70, 219-228 JONES R. (1998): Plasma membrane structure and remodelling during sperm maturation in the epididymis. J. Reprod. Fertil. Suppl. 53, 73-84, Review JONES R. and T. MANN (1973): Lipid peroxidation in spermatozoa. Proc. R. Soc. Lond. B. Biol. Sci. 184, 103–107 JONES R., T. MANN and R. SHERINS (1979): Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil. Steril. 31, 531-537 JOSHI M.S. (1988): Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow. J. Reprod. Fertil. 83, 249-261 JOSHI M.S. (1991): Growth and differentiation of the cultured secretory cells of the cow oviduct on reconstituted basement membrane. J. Exp. Zool. 260, 229-238 KANO K., T. MIYANO and S. KATO (1994): Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro. Theriogenology 42, 1061-1068
References
___________________________________________________________________
125
KARABINUS D.S., D.P. EVENSON, L.K. JOST, R.K. BAER and M.T. KAPROTH (1990): Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry. J. Dairy Sci. 73, 2364-2371 KATZ D.F. and R. YANAGIMACHI (1980): Movement characteristics of hamster spermatozoa within the oviduct. Biol. Reprod. 22, 759-764 KENNEY R.M., D.P. EVENSON, M.C. GARCIA and C.C. LOVE (1995): Relationships between sperm chromatin structure, motility, and morphology of ejaculated sperm, and seasonal pregnancy rate. Biol. Reprod. Monogr. 1, 647-653 KERVANCIOGLU M.E., O. DJAHANBAKHCH and R.J. AITKEN (1994): Epithelial cell coculture and the induction of sperm capacitation. Fertil. Steril. 61, 1103-1108 KESSOPOULOU E., M.J. TOMLINSON, C.L.R. BARRATT, A.E. BOLTON and I.D. COOKE (1992): Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J. Reprod. Fertil. 94, 463–470 KIEFER D., J.H. CHECK and D. KATSOFF (1996): The value of motile density, strict morphology, and the hypoosmotic swelling test in in vitro fertilization-embryo transfer. Arch. Androl. 37, 57-60 KING R.S., S.H. ANDERSON and G.J. KILLIAN (1994): Effect of bovine oviductal estrus-associated protein on the ability of sperm to capacitate and fertilize oocytes. J. Androl. 15, 468-478. KIM N.H., H. FUNAHASHI, L.R. ABEYDEERA, S.J. MOON, R.S. PRATHER and B.N. DAY (1996): Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro. J. Reprod. Fertil. 107, 79-86 KOOPMAN G., C.P. REUTELINGSPERGER, G.A. KUIJTEN, R.M. KEEHNEN, S.T. PALS and M.H. VAN-OERS (1994): Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415-1420
References
___________________________________________________________________
126
KOSOWER N.S., H. KATAYOSE and R. YANAGIMACHI (1992): Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei. J. Androl. 13, 342-348 KRAUSE D. (1965): Contributions to the andrology of the bull. Dtsch. Tierarztl. Wochenschr. 72, 454-458 KRUGER T.F., R. MENKVELD, F.S. STANDER, C.J. LOMBARD, J.P.VAN-DER-MERWE, J.A. VAN ZYL and K. SMITH (1986): Sperm morphologic features as a prognostic factor in in vitro fertilization. Ferti.l Steril. 46, 1118-1123 KULKARNI S.B., Z.E. SAUNA, V. SOMLATA and V. SITARAMAM (1997): Volume regulation of spermatozoa by quinine-sensitive channels. Mol. Reprod. Dev. 46, 535–550 KUMAROO K.K., G. JAHNKE and J.L. IRVIN (1975): Changes in basic chromosomal proteins during spermatogenesis in the mature rat. Arch. Biochem. Biophys. 168, 413-424 LANGLAIS J. and K.D. ROBERTS (1985): A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Research 12, 183-224 LAPOINTE S., R.C. CHIAN and M.A. SIRARD (1996): Effects of estrous cycle, steroids and localization of oviductal cells on in vitro secretion of sperm motility factor (s). Theriogenology 44, 119-128 LARSON-COOK K.L., J.D. BRANNIAN, K.A. HANSEN, K.M. KASPERSON, E.T. AAMOLD and D.P. EVENSON (2003): Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil. Steril. 80, 895-902 LARSSON B. and K. LARSSON (1985): Distribution of spermatozoa in the genital tract of artificially inseminated heifers. Acta Vet. Scand. 26, 385-395 LARSSON B. and K. LARSSON (1986): Sperm localization in the oviducts of artificially inseminated dairy cattle. Acta Vet. Scand. 27, 303-312
References
___________________________________________________________________
127
LARSSON B. and H. RODRIGUEZ-MARTINEZ (2000): Can we use in vitro fertilization tests to predict semen fertility? Anim. Reprod. Sci. 60-61, 327-336, Review LECHNIAK D., A. KEDZIERSKI and D. STANISLAWSKI (2002): The use of HOS test to evaluate membrane functionality of boar sperm capacitated in vitro. Reprod. Domest. Anim. 37, 379-380 LEESE H.J. (1988): The formation and function of oviduct fluid. J. Reprod. Fertil. 82, 843-856, Review LEFEBVRE R. (1997): The oviductal sperm reservoir in cattle and horses. Ph D. Thesis, Cornell Univ. Jan.1997 LEFEBVRE R., P.J. CHENOWETH, M. DROST, C.T. LE-CLEAR, M. MAC CUBBIN, J.T. DUTTON and S.S. SUAREZ (1995): Characterization of the oviductal sperm reservoir in cattle. Biol. Reprod. 53, 1066-1074 LEFEBVRE R., M.C. LO and S.S. SUAREZ (1997): Bovine sperm binding to oviductal epithelium involves fucose recognition. Biol. Reprod. 56, 1198-1204 LEFEBVRE R. and J.C. SAMPER (1993): A study of the interaction between stallion spermatozoa and oviductal explants in vitro. Equine Vet. J. (suppl 15), 39–41 LEFEBVRE R. and S.S. SUAREZ (1996): Effect of capacitation on bull sperm binding to homologous oviductal epithelium. Biol. Reprod. 54, 575-582 LEIDING C. (1996): Individual differences of ejaculates used for fresh semen AI Proc. Third Int. Conf. Boar Semen Preservation Reprod. Dom. Anim. 31, 121-128 LEMASTERS G.K., D.M. OLSEN, J.H.YIIN, J.E. LOCKEY, R. SNUKLA, S.G. SELEVAN, S.M. SCHRADER, G.P. TOTH, D.P. EVENSON and G.B. HUSZAR (1999): Male reproductive effects of solvent and fuel exposure during aircraft maintenance. Reprod. Toxicol. 13, 155-166
References
___________________________________________________________________
128
LINDNER, G.M., and R.O. WRIGHT (1983): Bovine embryo morphology and evaluation. Theriogenology, 20, 407-411 LIU D.Y., G.N. CLARKE and H.W. BAKER (1991): Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in vitro. J. Androl. 12, 231-239 LIU D.Y. and H.W. BAKER (1992): Sperm nuclear chromatin normality: relationship with sperm morphology, sperm zona pellucida binding, and fertilization rates in vitro. Fertil. Steril. 58, 1178-1184 LÖHMER I. (2003): Stabilität der Chromatinstruktur von Bullenspermien in Beziehung zu der konventionellen Spermatologie, der Bindungsfähigkeit im Ovidukt-Explant-Assay und der Fruchtbarkeit in vitro. School of veterinary, HANNOVER /GERMANY Thesis LONGO F.J., G. KROHNE and W.W. FRANKE (1987): Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements. J. Cell Biol. 105, 1105-1120 LOPES S., J.G. SUN, A.JURISICOVA, J. MERIANO and R.F. CASPER (1998): Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil. Steril. 69, 528–532 LOVE C.C. and R.M. KENNEY (1998): The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 50, 955-972 Erratum in: Theriogenology 51, 1207 LOVE C.C. and R.M. KENNEY (1999): Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol. Reprod. 60, 615-620 Erratum in: Biol. Reprod. 60, 1271 MACLEOD I.C. and D.S. IRVINE (1995): The predictive value of computer-assisted semen analysis in the context of a donor insemination programme. Hum. Reprod. 10, 580-658
References
___________________________________________________________________
129
MAGNUS F.K. (2002): Untersuchung der in vitro Bindungsfähigkeit porciner Spermien im Ovidukt Explant-Assay und im DudFinder® Sperm-Binding Assay und deren Beziehung zu spermatologischen Qualitätsparametern. School of veterinary, HANNOVER /GERMANY Thesis MAHADEVAN, M. M. and A. O., TROUNSON (1984): Relationship of fine structure of sperm head to fertility of frozen human semen. Fert. Steril. 41, :287-293 MAHMOUD A.I. and J.J. PARRISH (1996): Oviduct fluid and heparin induce similar surface Changes in bovine sperm during capacitation: a flow cytometric study using lectins. Mol. Reprod. Dev. 43, 554-560 MANICARDI G.C., P.G. BIANCHI, S. PANTANO, P. AZZONI, D. BIZZARO, U. BIANCHI and D. SAKKAS (1995): Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol. Reprod. 52, 864-867 MANSOUR R.T., M.A. ABOULGHAR, G.I. SEROUR, A.M. ABBAS, A.M. RAMZY and B. RIZK (1993): In vivo survival of spermatozoa in the human fallopian tube for 25 days: a case report. J. Assist. Reprod. Genet. 10, 379-380 MARTIN S.J., C.P. REUTELINGSPERGER, A.J. MC-GAHON, J.A. RADER, R.C. VAN-SCHIE, D.M. LA-FACE and D.R. GREEN (1995): Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545-1556 MATSUDA Y., I. TOBARI and T. YAMADA (1985): Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. II. Chromosome aberrations induced in mature oocytes and fertilized eggs at the pronuclear stage following X-irradiation. Mutat. Res. 151, 275-280 MAXWELL W.M.C. and L.A. JOHNSON (1997): Membrane status of boar spermatozoa after cooling or cryopreservation Theriogenology 48, 209–219
References
___________________________________________________________________
130
MAYER D.T., C.D. SQUIERS, R. BOGART and M.M. OLOUFA (1951): The technique for characterizing mammalian spermatozoa as dead or living by differential staining. J Anim Sci. 10, 226–235 MBURU J.N., S. EINARSSON, N. LUNDEHEIM and H. RODRIGUEZ-MARTINEZ (1996): Distribution, number and membrane integrity of spermatozoa in the pig oviduct in relation to spontaneous ovulation. Anim. Reprod. Sci. 45,109-121 MCCLURE R.D. and R. TOM (1991): Human sperm hypo-osmotic swelling test: relationship to sperm fertilizing ability. Int. J. Fertil. 36, 360-636 MC NUTT T.L. and G.J. KILLIAN (1991): Influence of bovine follicular and oviduct fluids on sperm capacitation in vitro. J. Androl. 12, 244-252 MC NUTT T.L., P. OLDS-CLARKE, A.L. WAY, S.S. SUAREZ and G.J. KILLIAN (1994): Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro. J. Androl. 15, 328-336 MEISTRICH M.L., W.A. BROCK, S.R. GRIMES, R.D. PLATZ and L.S. HNILICA (1978): Nuclear protein transitions during spermatogenesis. Fed. Proc. 37, 2522-2525, Review MEISTRICH M.L., B.O. REID and W.J. BARCELLONA (1976): Changes in sperm nuclei during spermiogenesis and epididymal maturation. Experimental Cell Research 99, 72–78 MENGHI G., A.M. BONDI, D. ACCILI, L. FUMAGALLI and G. MATERAZZI (1985): Characterisation in situ of the complex carbohydrates in rabbit oviduct using digestion with glycosidases followed by lectin binding. J. Anat. 140, 613-625 MENGHI G., P. SCOCCO and G. MATERAZZI (1995): Lectin binding and identification of sialic acid acceptor sugars in rabbit oviduct under hormone administration. Microsc. Res. Tech. 31, 488-496
References
___________________________________________________________________
131
MILLER O.C. and A.H. BLACKSHAW (1968): The DNA of rabbit spermatozoa aged in vitro and its relation to fertilization and embryo survival. In: Proceedings of the Vth International Congress of Animal Reproduction and Artificial Insemination, Paris, Vol II, p. 1275 MITCHELL J.R., P.L. SENGER and J.L. ROSENBERGER (1985): Distribution and retention of spermatozoa with acrosomal and nuclear abnormalities in the cow genital tract. J. Anim. Sci. 61, 956-967 MORALES P., V. PALMA, A.M. SALGADO and M. VILLALON (1996): Sperm interaction with human oviductal cells in vitro. Hum. Reprod. 11, 1504-1509 MORDEL N., S. MOR-YOSEF, E. MARGALIOTH, A. SHEMESH, A. SAMUELOFF and J.G. SCHENKER (1989): The human sperm hypoosmotic swelling test: its practical application and suggestions for improvement. Int. J. Fertil. 34, 355–358 MORRIS I.D., S. ILOTT, L. DIXON and D.R. BRISON (2002): The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum. Reprod. 17, 990-998 MORTIMER D., E.F. CURTIS and A.R. CAMEZIND (1990) Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa. Hum. Reprod. 5, 99–103 MORTIMER D., E.E. LESLIE, R.W. KELLY and A.A. TEMPLETON (1982): Morphological selection of human spermatozoa in vivo and in vitro. J. Reprod. Fertil. 64, 391-399 MOSSAD H., M. MORSHEDI, J.P. TONER and S. OEHNINGER (1994): Impact of cryopreservation on spermatozoa from infertile men: implications for artificial insemination. Arch. Androl. 33, 51-57 NAGAI T. and R.M. MOOR (1990): Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Mol. Reprod. Dev. 26, 377-382
References
___________________________________________________________________
132
NASR-ESFAHANI M.H., S. RAZAVI and M. MARDANI (2001): Relation between different human sperm nuclear maturity tests and in vitro fertilization. J. Assist. Reprod. Genet. 18, 219-225 NEILD D.M., M.G. CHAVES, M. FLORES, M.H. MIRAGAYA, E. GONZALEZ and A. AGUERO (2000): The HOS test and its relationship to fertility in the stallion. Andrologia 32, 351-355 NEILD D.M., M.G. CHAVES, M. FLORES, N. MORA, M. BECONI and A. AGUERO (1999): Hypoosmotic test in equine spermatozoa. Theriogenology 51, 721-727 ODOR D.L., P.GADDUM-ROSSE, R.E. RUMERY and R.J. BLANDAU (1980): Cyclic variations in the oviuductal ciliated cells during the menstrual cycle and after estrogen treatment in the pig-tailed monkey, Macaca nemestrina. Anat. Rec. 198, 35-57 OKADA A., K. INOMATA, M. MATSUHASHI, K. FUJIO, K. MIURA, K. SHIINA and M. SHIRAI (1990): Correlation of the hypo-osmotic swelling test, semen score and the zona-free hamster egg penetration assay in humans. Int. J. Androl. 13, 337-343 ORIHUELA P.A., M.E. ORTIZ and H.B. CROXATTO (1999): Sperm migration into and through the oviduct following artificial insemination at different stages of the estrous cycle in the rat. Biol. Reprod. 60, 908-913 OSTERHUIS G.J., R.M. HAMPSINK, H.W. MICHGELSEN and I. VERMES (1996): Hypo-osmotic swelling test: a reliable screening assay for routine semen specimen quality screening. J. Clin. Lab. Anal. 10, 209-212 OUHIBI N., Y. MENEZO, G. BENET and B. NICOLLET (1989): Culture of epithelial cells derived from the oviduct of different species. Hum. Reprod. 4, 229-235 OVERSTREET J.W. and G.W. COOPER (1975): Reduced sperm motility in the isthmus of the rabbit oviduct. Nature 258, 718-719
References
___________________________________________________________________
133
OVERSTREET J.W. and G.W. COOPER (1978a): Sperm transport in the reproductive tract of the female rabbit: I. The rapid transit phase of transport. Biol. Reprod. 19, 101-114 OVERSTREET J.W. and G.W. COOPER (1978b): Sperm transport in the reproductive tract of the female rabbit: II. The sustained phase of transport. Biol. Reprod. 19, 115-132 OVERSTREET L.W. and D.F. KATZ (1977): Sperm transport and selection in the female genital tract. In: JOHNSON MH (ed) Development in Mammals, volume 2. Amesterdam: Elsevier, pp31-65 PACEY A.A., C.J. HILL, I.W. SCUDAMORE, M.A. WARREN, C.L. BARRATT and I.D. COOKE (1995): The interaction in vitro of human spermatozoa with epithelial cells from the human uterine (fallopian) tube. Hum. Reprod. 10, 360-366 PARKER W.G., J.J. SULLIVAN and N.L. FIRST (1975): Sperm transport and distribution in the mare. J. Reprod. Fertil. Suppl. (23): 63-66 PARRISH J.J., J. SUSKO-PARRISH, M.A. WINER and N.L. FIRST (1988): Capacitation of bovine sperm by heparin. Biol Reprod. 38,1171-1180. PARRISH J.J., J.L. SUSKO-PARRISH, R.R. HANDROW, M.M. SIMS and N.L. FIRST (1989): Capacitation of bovine spermatozoa by oviduct fluid. Biol. Reprod. 40, 1020-1025 PARRISH J.J., A. KROGENAES, and L. SUSKO-PARRISH (1995): Effect of bovine sperm separation by either swim-up or percoll method on success of in vitro fertilization and early embryonic development. Theriogenology 44, 859-869 PENTIKAINEN V., K. ERKKILA and L. DUNKEL (1999): Fas regulates germ cell apoptosis in the human testis in vitro. Am. J. Physiol. 276, 310-316 PEREZ-LLANO B., J.L. LORENZO, P. YENES, A. TREJO and P. GARCIA-CASADO (2001): A short hypoosmotic swelling test for the prediction of boar sperm fertility. Theriogenology 56, 387-398
References
___________________________________________________________________
134
PERREAULT S.D., J. RUBES, W.A. ROBBINS, D.P. EVENSON and S.G. SELEVAN (2000): Evaluation of aneuploidy and DNA damage in human spermatozoa: application in field studies Andrologia 32, 247-254 PETRUNKINA A.M., J. FRIEDRICH, W. DROMMER, G. BICKER, D. WABERSKI and E. TÖPFER-PETERSEN (2001a): Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+ concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells. Reproduction 122, 469-480 PETRUNKINA A.M, HEBEL M., D. WABERSKI, K. F. WEITZE and E. TÖPFER-PETERSEN (2004): Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa. Reproduction. 127, 105-115 PETROUNKINA A. M., R. A. P. HARRISON, R.PETZOLDT, K. F WEITZE. and E. TÖPFER-PETERSEN (2000): Cyclical changes in sperm volume during in vitro incubation under capacitating conditions: a novel boar sperm characteristic. J. Reprod. Fertil. 118, 283–293. PETRUNKINA A.M., R. GEHLHAAR, W. DROMMER, D. WABERSKI and E. TÖPFER-PETERSEN (2001b): Selective sperm binding to pig oviductal epithelium in vitro. Reproductionn 121, 889-896 PETRUNKINA A.M., R. PETZOLDT, S. STAHLBERG, J. PFEILSTICKER, M. BEYERBACH, H. BADER and E. TÖPFER-PETERSEN (2001c): Sperm-cell volumetric measurements as parameters in bull semen function evaluation: correlation with no return rate. Andrologia 33, 360-367 PETRUNKINA A.M., R. A. HARRISON, M. HEBEL, K. F. WEITZE and E. TÖPFER-PETERSEN (2001d): Role of quinine-sensitive ion channels in volume regulation in boar and bull spermatozoa. Reproduction 122, 327-336 PETRUNKINA A.M. and E. TÖPFER-PETERSEN (2000): Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane. Reprod. Fertil. Dev. 12, 297-305
References
___________________________________________________________________
135
PETZOLDT.R. (1988): Elektronische Volumenanalyse: Eine Methode zur Beurteilung DER Intaktheit DER Spermienzellmembran. Wissenschaftliche Zeitschrift Humboldt-Universität zu Berlin, Reihe Medizin 37, 121–124 PETZOLDT.R. and S. ENGEL (1994): The spermatozoal volume as indicative of the plasma membrane integrity (modification of the hypoosmotic swelling test) II: Diagnostic approach. Andrologia 26, 315-321 PIRHONEN A., A. LINNALA-KANKKUNEN and P.H. MAENPAA (1994): Identification of phosphoseryl residues in protamines from mature mammalian spermatozoa. Biol. Reprod. 50, 981-986 POGANY G.C., M. CORZETT, S. WESTON and R. BALHORN (1981): DNA and protein content of mouse sperm, Implications regarding sperm chromatin structure Exp. Cell Res. 136, 127-136 POLLARD J.W. (1992): Functional biology of bovine oviductal epithelial cells in vitro. Guelph, Canada: University of Guelph; 1992. Thesis 13:127–139 POLLARD J.W., C. PLANTE, W.A. KING, P.J HANSEN, K.J. BETTERIDGE and S.S. SUAREZ (1991): Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells. Biol. Reprod. 44, 102-107 PRIETO M.C., A.H. MAKI and R. BALHORN (1997): Analysis of DNA-protamine interactions by optical detection of magnetic resonance. Biochemistry 36, 11944-11951 RACY P.A. (1975): The prolonged survival of spermatozoa in bats, in: DUCKETT JG, RACYPA (ed) Biology of the female gamete. London: Academic Press, pp.385-416 RAMIREZ J.P., A. CARRERAS and C. MENDOZA (1992): Sperm plasma membrane integrity in fertile and infertile men. Andrologia 24, 141-144 RAYCHOUDHURY S.S. and S.S. SUAREZ (1991): Porcine sperm binding to oviductal explants in culture. Theriogenology 36, 1059 -1070
References
___________________________________________________________________
136
RAYCHOUDHURY S.S., S.S. SUAREZ and W.C. BUHI (1993): Distribution of lectin binding sites in the oviducts of cycling and hormone-treated pigs. J. Exp. Zool. 265, 659-668 REVAH I., B.M. GADELLA, F.M. FLESCH, B. COLENBRANDER and S.S. SUAREZ (2000): Physiological state of bull sperm affects fucose- and mannose-binding properties. Biol. Reprod. 62, 1010 -1015 RIGLER R J.R. (1966): Microfluorometric characterization of intracellular nucleic acids and nucleoproteins by acridine orange. Acta Physiol. Scand. Suppl. 267, 1-122 RODRIGUEZ-GIL J.E., A. MONTSERRAT and T. RIGAU (1994): Effects of hypo-osmotic incubation on acrosome and tail structure on canine spermatozoa. Theriogenology 42, 815-829 RODRIGUEZ H., C. OHANIAN and E. BUSTOS-OBREGON (1985): Nuclear chromatin decondensation of spermatozoa in vitro: a method for evaluating the fertilizing ability of ovine semen. Int. J. Androl. 8, 147-158. RODRIGUEZ-MARTINEZ, H. and A. BERROSTEGUIETA (1994): Viability of frozen-thawed bull spermatozoa after swim-up through a Hyaluronic acid solution. Biomedicine Research 2, 133 144. RODRIGUEZ-MARTINEZ H., B. LARSSON and H. PERTOFT (1997): Evaluation of sperm damage and techniques for sperm clean up. Reprod. Fertil. Dev. 9, 297-308. Review ROSCHLAU G. (1965): Contribution to the histochemical behavior of nuclear DNA in the interphase and mitosis, demonstrated by combined acid hydrolysis and acridine orange fluorescent chromatization]. Histochemie. 5, 396-406, German ROTA A., N. PENZO, L. VINCENTI and R. MANTOVANI (2000): Hypoosmotic swelling (HOS) as a screening. Theriogenology 53, 1415-1420 ROUX C and J.P. DADOUNE (1989): Use of acridine orange staining on smears of human spermatozoa after heat- treatment: evaluation of the chromatin condensation. Andrologia 21, 275-280
References
___________________________________________________________________
137
ROYERE D., S. HAMAMAH, J.C.NICOLLE, C. BARTHELEMY and J. LANSAC (1988): Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Feulgen-DNA cytophotometric studies. Gamete Res. 21, 51-57 ROYERE D., S. HAMAMAH, J.C. NICOLLE and J. LANSAC (1991a): Chromatin alterations induced by freeze-thawing influence the fertilizing ability of human sperm. Int. J. Androl. 14, 328-332 ROYERE D., S. HAMAMAH, J.C. NICOLLE and J. LANSAC (1991b): Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? Cytochemical studies. Mol. Reprod. Dev. 28, 177-182 RUKNUDIN A. and I.A. SILVER (1990): Ca2+ uptake during capacitation of mouse spermatozoa and the effect of an anion transport inhibITOr on Ca2+ uptake. Mol. Reprod. Dev. 26, 63-68 SAILER B.L., L.K. JOST and D.P. EVENSON (1996): Bull sperm head morphometry related to abnormal chromatin structure and fertility. Cytometry. 24, 167-173 SAKKAS D., E. MARIETHOZ, G. MANICARDI, D. BIZZARO, P.G. BIANCHI and U. BIANCHI (1999): Origin of DNA damage in ejaculated human spermatozoa. Rev. Reprod. 4, 31-37, Review SAKKAS D., F. URNER, P.G. BIANCHI, D. BIZZARO, I. WAGNER, N. JAQUENOUD, G. MANICARDI and A. CAMPANA (1996): Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum. Reprod. 11, 837-843 SALISBURY G.W. and R.G. HART (1970): Gamete aging and its consequences. Biol. Reprod. Suppl. 2, 1-13 SALLAM H.N., F. EZZELDIN, A. SALLAM, A.F. AGAMEYA and A. FARRAG (2003): Sperm velocity and morphology, female characteristics, and the hypo osmotic swelling test as predictors of fertilization potential: experience from the IVF model. Int. J Fertil. Womens Med. 48, 88-95
References
___________________________________________________________________
138
SATOH T., H. ABE, Y. SENDAI, H. IWATA and H. HOSHI (1995): Biochemical characterization of a bovine oviduct-specific sialo-glycoprotein that sustains sperm viability in vitro. Biochim. Biophys. Acta. 1266, 117-123 SCHULTE B.A., K.P. RAO, A. KREUTNER, G.N. THOMOPOULOS and S.S. SPICER (1985): Histochemical examination of glycoconjugates of epithelial cells in the human fallopian tube. Lab. Invest. 52, 207-219 SCOTT M.A., I.K.M. LIU and J.W. OVERSTREET (1995): Sperm transport to the oviducts: Abnormalities and their clinical implications. Proceedings of the Annual AAEP Convention 41: 1-2 SELEVAN S.G., L. BORKOVEC, V.L. SLOTT, Z. ZUDOVA, J. RUBES, D.P. EVENSON and S.D. PERREAULT (2000): Semen quality and reproductive health of young czech men exposed to seasonal air pollution. Environ. Health Perspect. 108, 887-894 SHALET S.M. (1980): Effect of cancer chemotherapy on gonadal function of patients. Cancer Treat. Rev. 7, 141-152, Review SHALGI R. and P.F. KRAICER (1978): Timing of sperm transport, sperm penetration and cleavage in the rat. J. Exp. Zool. 204, 353-360 SHALGI R., T.T. SMITH and R. YANAGIMACHI (1992): A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction. Biol. Reprod. 46, 419-424 SHANNON P. and B. CURSON (1972): Toxic effect and action of dead sperm on diluted bovine semen. J. Dairy Sci. 55, 614–620. SHANNON P. and B. CURSON (1981): Site of aromatic L-amino acid oxidase in dead bovine spermatozoa and determination of between-bull differences in the percentage of dead spermatozoa by oxidase activity. J. Reprod. Fertil. 64, 469–473.
References
___________________________________________________________________
139
SHANNON P. and B. CURSON (1982): Kinetics of the aromatic L-amino acid oxidase from dead bovine spermatozoa and the effect of catalase on fertility of diluted bovine semen stored at 5°C and ambient temperatures. J. Reprod. Fertil. 64, 463–470 SHEN H.M., S.E. CHIA and C.N. ONG (1999): Evaluation of oxidative DNA damage in human sperm and its association with male infertility. J. Androl. 20, 718-723 SINGH J.P., D.F. BABCOCK and H.A. LARDY (1978): Increased calcium-ion influx is a component of capacitation of spermatozoa. Biochem. J. 172, 549-556 SJOBLUM P. and E. COCCIA (1989): On the diagnostic value of the hypoosmotic sperm swelling test in an in vitro fertilization program. J. In vitro Fertil. Embryo Transfer 6, 41–43 SMITH R., M. MADARIAGA and E. BUSTOS-OBERGON (1992): Reappraisal of the hypo-osmotic swelling test to improve assessment of seminal fertility status. Int. J. Androl. 15, 5-13 SMITH T.T. (1998): The modulation of sperm function by the oviductal epithelium. Biol. Reprod. 58, 1102-1104, Review SMITH T.T., F. KOYANAGI and R. YANAGIMACHI (1987): Distribution and number of spermatozoa in the oviduct of the GOLDen hamster after natural mating and artificial insemination. Biol. Reprod. 37, 225-234 SMITH T.T. and R. YANAGIMACHI (1989): Capacitation status of hamster spermatozoa in the oviduct at various times after mating. J. Reprod. Fertil. 86, 255-261 SMITH T.T. and R. YANAGIMACHI (1990): The viability of hamster spermatozoa stored in the isthmus of the oviduct: the importance of sperm-epithelium contact for sperm survival. Biol. Reprod. 42, 450-457
References
___________________________________________________________________
140
SMITH T.T. and R. YANAGIMACHI (1991): Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct. J. Reprod. Fertil. 91, 567-573 SPANO M., E. CORDELLI, G. LETER, F. LOMBARDO, A. LENZI and L. GANDINI (1999): Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay. Mol. Hum. Reprod. 5, 29-37 SPANO M., A.H. KOLSTAD, S.B. LARSEN, E. CORDELLI, G. LETER, A. GIWERCMAN and J.P. BONDE (1998): The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies, Asclepios. Hum. Reprod. 13, 2495-2505 STALHAMMAR E.M., L. JANSON and J. PHILIPSSON (1994): The impact of sperm motility on non-return rate in preselected dairy bulls. Reprod. Nutr. 34, 37-45 STALHEIM O.H., J.E. GALLAGHER and B.L. DEYOE (1975): Scanning electron microscopy of the bovine, equine, porcine, and caprine uterine tube (oviduct). Am. J. Vet. Res. 36, 1069-1075 STONE B.A. (1977): Between and within herd variations in conception rates in pig herds in the lower North of South Australia. Agric. Rec. S. Aust. 4, 22-25 STRANGE K., F. EMMA and P.S. JACKSON (1996): Cellular and molecular physiology of volume-sensitive anion channels. Am. J. Physiol. 270, C711–C730 SUAREZ S.S. (1987): Sperm transport and motility in the mouse oviduct: observations in situ. Biol. Reprod. 36, 203-210 SUAREZ S.S. (1996): Hyperactivated motility in sperm. J. Androl. 17, 331-335, Review SUAREZ S.S., X.B. DAI, R.P. DE-MOTT, K. REDFERN and M.A. MIRANDO (1992): Movement characteristics of boar sperm obtained from the oviduct or hyperactivated in vitro. J. Androl. 13, 75-80
References
___________________________________________________________________
141
SUAREZ S.S., M. DROST, K. REDFERN and W. GOTTLIEB (1990): Sperm motility in the oviduct, in: Bavister BD, CUMMINS J, Roldan ERS (eds.) Fertilization in Mammals. Norwell: Serono Symposia: 111–124 SUAREZ S.S. and R.A. OSMAN (1987): Initiation of hyperactivated flagellar bending in mouse sperm within the female reproductive tract. Biol. Reprod. 36, 1191-1198 SUAREZ S.S., K. REDFERN, P. RAAYNOR, F. MARTIN and D.M. PHILLIPS (1991): Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir. Biol. Reprod. 44, 998-1004 SUAREZ S.S., K. BROCKMAN and R. LEFEBVRE (1997): Distribution of mucus and sperm in bovine oviducts after artificial insemination: the physical environment of the oviductal sperm reservoir. Biol. Reprod. 56,447-453 SUAREZ S.S., I.REVAH, M. LO and S. KOLLE (1998): Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognizes LEWIS-a trisaccharide. Biol. Reprod. 59, 39-44 SUN J.G., A. JURISICOVA and R.F. CASPER (1997): Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol. Reprod. 56, 602–607 SUZUKI H. and R.H. FOOTE (1995): Bovine oviductal epithelial cells (BOEC) and oviducts: I. For embryo culture, II. Using SEM for studying interactions with spermatozoa. Microsc. Res. Tech. 31, 519-530 SUZUKI H., R.H. FOOTE and P.B. FARRELL (1997): Computerized imaging and scanning electron microscope (SEM) analysis of co-cultured fresh and frozen bovine sperm. J. Androl. 18, 217-226 SWANSON E.W. and H.J. BEARDEN (1951): An eosin/nigrosin stain for differentiating live and dead spermatozoa. J. Anim. Sci. 10, 981–987
References
___________________________________________________________________
142
TAGGART D.A. and P.D. TEMPLE-SMITH (1991): Transport and storage of spermatozoa in the female reproductive tract of the brown marsupial mouse, Antechinus stuartii (Dasyuridae). J. Reprod. Ferti. 93, 97-110 TAMULI M.K. and P.F. WATSON (1994): Use of a simple staining technique to distinguish acrosomal Changes in the live sperm sub-population Animal. Reprod. Sci. 35, 247–254 TAO J., E.S. CRISTER and J.K. CRISTER (1993): Evaluation of mouse sperm acrosomal status and viability by flowcytometry. Mol. Reprod. Dev. 36, 183 –194 TARTAGNI M., M.M.SCHONAUER, E. CICINELLI, H. SELMAN, D. DE-ZIEGLER, F. PETRUZZELLI and V. D'ADDARIO (2002): Usefulness of the hypo-osmotic swelling test in predicting pregnancy rate and outcome in couples undergoing intrauterine insemination. J. Androl. 23, 498-502 TEJADA R.I., J.C. MITCHELL, A. NORMAN, J.J. MARIK and S. FRIEDMAN (1984): A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil. Steril. 42, 87-91 TERQUEM, A. and J.P. DADOUNE (1983): Aniline BLUE staining of human spermatozoa chromatin: evaluation of nuclear maturation. In: J. ANDRÉ (Ed): The sperm cell. London: Martinus Nijhoff Publishers, pp. 249-252. THIBAULT C., T M.COURO, L. MARTINET, P. MAULEON, F D.U. MESNIL, D.U. BUISON, R. ORTAVANT, J. PELLETIER and J.P. SIGNORET (1966): Regulation of breeding season and estrous cycles by light and external stimuli in some mammals. J. Anim. Sci., Suppl 1, 25, 119-142 THOMAS P.G. and B.A. BALL (1996): Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro. Mol. Reprod. Dev. 43, 55-61 THOMAS P.G., B.A. BALL and S.P. BRINSKO (1994a): Interaction of equine spermatozoa with oviduct epithelial cell explants is affected by estrous cycle and anatomic origin of explant. Biol. Reprod. 51, 222-228
References
___________________________________________________________________
143
THOMAS P.G., B.A. BALL, P.G. MILLER, S.P. BRINSKO and L. SOUTHWOOD (1994b): A subpopulation of morphologically normal, motile spermatozoa attach to equine oviductal epithelial cell monolayers. Biol. Reprod. 51, 303-309 THOMAS P.G., B.A. BALL and S.P. BRINSKO (1995a): Changes Associated with Induced Capacitation Influence the Interaction between Equine Spermatozoa and Oviduct Epithelial Cell Monolayers. Biol. Reprod. Monograph 1, 697-705 THOMAS P.G., G.G. IGNOTZ, B.A. BALL, S.P. BRINSKO and W.B. CURRIE (1995b): Effect of coculture with stallion spermatozoa on de novo protein synthesis and secretion by equine oviduct epithelial cells. Am. J. Vet. Res. 56, 1657-1662 THOMAS P.G., G.G. IGNOTZ, B.A. BALL, P.G. MILLER, S.P. BRINSKO and B. CURRIE (1995c): Isolation, culture, and characterization of equine oviduct epithelial cells in vitro. Mol. Reprod. Dev. 41, 468-478 THUNDATHIL J., J. GIL, A. JANUSKAUSKAS, B. LARSSON, L. SODERQUIST, R. MAPLETOFT and H. RODRIGUEZ-MARTINEZ (1999): Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination. Int. J. Androl.. 22, 366-673 TÖPFER-PETERSEN E. (1999): Molecules on the sperm's route to fertilization. J. Exp. Zool. 285, 259-266, Review TWIGG J., N. FULTON, E. GOMEZ, D.S. IRVINE and R.J. AITKEN (1998): Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod. 13, 1429-1436 UCHIDA A., K. TAKAHASHI and M. KITAO (1992): Usefulness of the hypo-osmotic swelling test for evaluation of human sperm fertilization. Hum. Reprod. 7, 1264-1267
References
___________________________________________________________________
144
VALCARCEL A., M.A. DE-LAS-HERAS, L.J. PEREZ, D.F. MOSES and H. BALDASSARRE (1997): Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining. Anim. Reprod. Sci. 45, 299–309 VANDEN BOSCH, G. C. and E. S. HAFEZ (1974): Sperm transport and distribution in the reproductive tract of the female rabbit after intraperitoneal insemination. Fertil. Steril. 25, 1039-1046. VAN-DEN-SAFFELE J., L. VERMEULEN, F. SCHOONJANS and F.H. COMHAIRE (1992): Evaluation of the hypo-osmotic swelling test in relation with advanced methods of semen analysis. Andrologia 24, 213-217 VAN-DER-SCHANS G.P., R. HARING, H.C. VAN-DIJK-KNIJNENBURG, P.L. BRUIJNZEEL and N.H. DEN-DAAS (2000): An immunochemical assay to detect DNA damage in bovine sperm. J. Androl. 21, 250-257 VAN-DER-VEN H.H., R.S. JEYENDRAN, S. AL.-HASANI, M. PEREZ-PELAEZ, K. DIEDRICH and L.J.D. ZANEVELD (1986): Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization. J. Androl. 7, 190–196 VAN-LANGENDONCKT A., A. VANSTEENBRUGGE, C. DESSY-DOIZE, J.E. FLECHON, G. CHARPIGNY, P. MERMILLOD, A. MASSIP and F. DESSY (1995): Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions. In vitro Cell Dev. Biol. Anim. 31, 664-670 VAZQUEZ J.M., E.A. MARTINEZ, P. MARTINEZ, C. GARCIA-ARTIGA and J. ROCA (1997): Hypoosmotic swelling of boar spermatozoa compared to other methods for analysing the sperm membrane. Theriogenology 47, 913-922 VED S., M. MONTAG, A. SCHMUTZLER, G. PRIETL, G. HAIDL and H. VAN-DER-VEN (1997): Pregnancy following intracytoplasmic sperm injection of immotile spermatozoa selected by the hypo-osmotic swelling-test: a case report. Andrologia 29, 241-242
References
___________________________________________________________________
145
WABERSKI D.,I. LÖHMER, A. A. Y. KHALIL, M. BAYERBACH, and E. TÖPFER-PETERSEN (2003a): Preferential binding of chromatin-stable bull spermatozoa to oviductal epithelium in vitro. 7th Annual Conference for Domestic Animal Reproduction, Dublin 4-6 Sept. 2003 In Reprod. Dom. Anim. 38 (2003), 335-336 WABERSKI D, A. M PETRUNKINA, J. FRIEDRICH, F. MAGNUS, A.A.Y. KHALIL and E. TÖPFER-PETERSEN (2003b): In Vitro-Studies of Sperm Binding and Survival at Oviductal Epithelium in Pigs and Cattle. 7th Annual Conference for Domestic Animal Reproduction, Dublin 4-6 Sept. 2003 In Reprod. Dom. Anim. 38 (2003), 325 WAGNER A, M. EKHLASI-HUNDRIESER, C. HETTEL, A. PETRUNKINA, D. WABERSKI, M. NIMTZ and E. TÖPFER-PETERSEN (2002): Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig. Mol. Reprod. Dev. 61, 249-257 WANG C., S.Y.W. CHAN, M. NG, W.W. SO, W.L. TSOI, T. LO and J. LEUNG (1988): A. Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men. J. Androl. 9, 384–389 WANG W.H., L.R. ABEYDEERA, L.R. FRASER and K. NIWA (1995): Functional analysisusing chlortetRACYcline fluorescence and in vitro fertilization of frozen–thawed ejaculated boar spermatozoa incubated in a protein-free chemically defined medium. J. Reprod. Fertil. 104, 305–313 WARD W.S. (1993): Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol. Reprod. 48, 1193-1201, Review WARD W.S. and D.S. COFFEY (1991): DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol. Reprod. 44, 569-574, Review WARRANT R.W. and S.H. KIM (1978): Alpha-Helix-double helix interaction shown in the structure of a protamine-transfer RNA complex and a nucleoprotamine model. Nature 1271, 130-135
References
___________________________________________________________________
146
WATSON P.F. (1995): Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871–891 WHYTE A., C. YANG, F. RUTTER and R.B. HEAP (1987): Lectin-binding characteristics of mouse oviduct and uterus associated with pregnancy block by autologous antiprogesterone monoclonal antibody. J. Reprod. Immunol. 11, 209 -219 WILLIAMS M., C.J. HILL, I.SCUDAMORE, B. DUNPHY, I.D. COOKE and C.L. BARRATT (1993): Sperm numbers and distribution within the human fallopian tube around ovulation. Hum. Reprod. 8, 2019-2026 WILMUT I. and R.H. HUNTER (1984): Sperm transport into the oviducts of heifers mated early in oestrus. Reprod. Nutr. Dev. 24, 461-468 WOLDESENBET S. and G.R. NEWTON (2003): Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro. Theriogenology 60, 533-543 WRENZYCKI, C. (1995): Experimentelle Untersuchungen zur Expression des Gap junction-Gens Konnexin43 in in vivo und in vitro produzierten präimplantatorischen Rinderembryonen mit Hilfe der Reversen Transkriptase Polymeraseketten-reaktion (RT-PCR). School of veterinary, HANNOVER /GERMANY, Thesis WU A.S., S.D. CARLSON and N.L. FIRST (1976): Scanning electron microscopic study of the porcine oviduct and uterus. J. Anim. Sci. 42, 804-809 WU T.C., S.M. LEE, M.H. JIH, J.T. LIU and Y.J. WAN (1993): Differential distribution of glycoconjugates in human reproductive tract Fertil Steril. 59, 60-64 WYLLIE A.H. (1980): Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556
References
___________________________________________________________________
147
WYROBEK, A.J., S.M. SCHRADER, S.D. PERREAULT, L. FENSTER, G. HUSZAR, D.F. KATZ, A.M. OSORIO, V. SUBLET and D.P. EVENSON (1997): Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites, III. Guidelines for field studies of male reproductive disorders. Reprod. Toxicol. 11, 243-259 YANAGIMACHI R. (1994): Fertility of mammalian spermatozoa: its development and relativity. Zygote 2, 371-2, Review YANAGIMACHI R. and C.A. MAHI (1976): The sperm acrosome reaction and fertilization in the guinea pig: a study in vivo. J. Reprod. Fertil. 46, 49-54 YAVETZ H, R. HAUSER, L. YOGEV, A. BOTCHAN, J.B. LESSING, Z.T. HOMONNAI and G. PAZ (1995): Advanced methods for evaluation of sperm quality. Andrologia 27, 31-35 YEUNG W.S., V.K. NG, E.Y. LAU and P.C. HO (1994): Human oviductal cells and their conditioned medium maintain the motility and hyperactivation of human spermatozoa in vitro. Hum. Reprod. 9, 656-660 YEUNG C.H., E. SONNENBERG-RIETHMACHER and T.G. COOPER (1999): Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms. Biol. Reprod. 61, 1062-1069 YEUNG C.H., A. WAGENFELD, E. NIESCHLAG and T.G. COOPER (2000): The cause of infertility of male c-ros tyrosine kinase receptor knockout mice. Biol. Reprod. 63, 612-618 ZAMBONI L. (1972): Fertilization in the mouse in:Moghissi KS, HAFEZ ESE (eds.), Biolohgy of mammalian fertilization. Springfield, IL: CHARLES C. THOMAS; 213-262 ZEYNELOGLU H.B., V. BALTACI, S. EGE, A. HABERAL and S. BATIOGLU (2000): Detection of chromosomal abnormalities by fluorescent in-situ hybridization in immotile viable spermatozoa determined by hypo-osmotic sperm swelling test. Hum. Reprod. 15, 853-856
References
___________________________________________________________________
148
ZHANG B.R., B. LARSSON, N. LUNDEHEIM, M.G. HAARD and H. RODRIGUEZ-MARTINEZ (1999): Prediction of bull fertility by combined in vitro assessments of frozen-thawed semen from young dairy bulls entering an AI-programme. Int. J. Androl. 22, 253-260 ZHANG BR, B. LARSSON, N. LUNDEHEIM and H. RODRIGUEZ-MARTINEZ (1998): Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls. Int. J. Androl. 21, 207-216 ZHU J., C.L. BARRATT, J. LIPPES, A.A. PACEY, E.A. LENTON and I.D. COOKE (1994): Human oviductal fluid prolongs sperm survival. Fertil. Steril. 61, 360-366 ZISKIND G., Y. PALTIELI, I. EIBSCHITZ, G. OHEL and A. WEICHSELBAUM (2000): The effect of human fallopian tube epithelium on human sperm velocity motility and binding. J. Assist. Reprod. Genet. 17, 147-150
Appendix
___________________________________________________________________
149
9 APPENDIX
9.1 LABORATORY NEEDS
-Dist. Water Preparation system,
(Umkehr-Osmose-Anlage)
-Slides (76 x 26 mm)
-Cover slides (50 x 24 mm) and
(18 x 18 mm)
-Disposable filter, 0,2 µm (Minisart®)
-Eppendorf-Reaction tubes, 1,5 ml
-Fluorescence microscope with phase
Contrast equipment
-Glass centrifuge tubes with conical bottom
-Refrigerator
-Laboratory bottles with thread, 250, 1000 ml
-Laboratory balance, Typ L310
-Laboratory centrifuge (Megafuge 2.0 R)
-Parafilm®
-Phase contrast microscope with warm stage
-Phase contrast microscope without warm stage
-Pipettes, adjustable (automatic pipettes)
(100-1000 µl, 20-200 µl, 1-20 µl)
-Tips of pipettes (blue and yellow)
-Warm plate (Hotplate SH 2 D)
-Water bath with temperature control
-Water-operated vacuum pump (Haake)
-Counting chamber
-Co. Kloos,Langenhagen, HANNOVER
-Community of interests of the laboratory
specialist trader GmbH & Co KG (IDL),
Nidderau
-Co. Sartorius GmbH, Göttingen
-Co. Greiner, Frickenhausen
-Co. Zeiss (Axioskop), Jena
-Co. Sarstedt, Nümbrecht
-Co. Liebherr, Nürnberg
-Co. Jürgens (9.072 0025), (9.072 010),
HANNOVER
-Co. Sartorius GmbH, Göttingen
-Co. Heraeus Instruments, Hamburg
-Co. American National Can™, USA
-Co. Zeiss (1697), Jena
-Co. Zeiss, Jena
-Co. Abimed, Langenfeld
-Co Greiner (685290und686290),
Frickenhausen
-Co Stuart Scientific, England
-Co for laboratories GmbH (GFL),Burgwedel
-Co Landgraf Laborgeräte, Langenhagen
-Thoma Co. Glass wares Hecht, Sontheim
Appendix
___________________________________________________________________
150
9.2 CHEMICALS AND REAGENTS
-BSA (Cohn’s Fraktion V)
-Calciumchloride (CaCl2)
-EDTA
-Gentamycinsulfate
-Glucose
-Hepes
-Kaliumchloride (KCl)
-Kaliumhydroxide (KOH)
-Kaliumdihydrogenphosphate (KH2PO4)
-Magnesiumchloride (MgCl2)
-Natriumbikarbonate (NaHCO3)
-Natriumchloride (NaCl)
-Natriumcitrate
-Natriumlactate
-Natriumdihydrogenphosphat (NaH2PO4)
-Dinatriumhydrogenphosphat (Na2HPO4)
-Natriumpyruvate
-Percoll®
-Propidiumiodide (PI) & SYBR14
-Streptomycine (Strepto-Hefa)
-tri-Natrium citrate
-Ethanol (96%)
-Co. Merck (1.12018.0025), Darmstadt
-Co. Merck (1.02382.0500), Darmstadt
-Co. Merck (1.08418.0100), Darmstadt
-Co. Serva (22185.02), Heidelberg
-Co. Merck (1.04074.0500), Darmstadt
-Co. Merck (1.10110.0250), Darmstadt
-Co. Merck (1.04938.0500), Darmstadt
-Co. Merck (1.05033.0500), Darmstadt
-Co. Merck (1.04873.0250), Darmstadt
-Co. Merck (1.05833.1000), Darmstadt
-Co. Merck (1.06329.0500), Darmstadt
-Co. Roth (9265.2), Karlsruhe
-Co. Merck (1.06448.0500), Darmstadt
-Co. Fluka AG, Schweiz
-Co. Merck (1.06346.0500), Darmstadt
-Co. Merck (1.06586.0500), Darmstadt
-Co. Merck (1.06619.0500), Darmstadt
-Co. Sigma-Aldrich (P-1644),Deisenhofen
-Co. Calbiochem Novabiochem GmbH,
(537059), Bad Soden / Taunus Molecular
Probes, Mol. Biol. Tech., Göttingen
-Co. Hefa Pharm GmbH & Co KG, Werne
-Co. Merck (1.06448.1000), Darmstadt
-Co. CG Chemicals, Laatzen
Appendix
___________________________________________________________________
151
9.3 BUFFERS, SOLUTIONS, MEDIA AND DILUENTS
9.3.1 Natrium chloride solution 10 % (for killing of spermatozoa)
- 10 gm NaCl /100 ml D.W. (to kill spermatozoa for estimation of sperm cell
density
9.3.2 Formolcitrat (for fixation of spermatozoa)
4 parts of 37 % formaldehyde solutions are mixed with 96 parts of a 2.9 %
Natrium citrate solution. This solution is suited to the liquid (wet) fixation of
spermatozoa.
9.3.3 Hepes buffer saline (HBS) for m-HOST
a) 300 mOsmol (isotonic solution):
- NaCl 137.0 mM (8.0 g / L)
- Glucose 10.0 mM (1.8 g / L)
- Hepes 20 mM (4.77 g / L)
- KOH 2.5 mM (2.5 ml 1M / L)
b) 180 mOsmol (hypotonic solution): - 600 ml HBS isotonic (300 mOsmol) + 400 ml dist. water (0 mOsmol)
- Both iso- and hypo-tonic solutions were sterile filtered before use.
9.3.4 Percoll® Solution (after HARRISON et al., 1993)
1. 10-times concentrated stock solution of Hepes Saline
-NaCl 137 mM (8.0 g /100ml)
-Hepes 20 mM (4.77 g/100 ml)
-Glucose x H2O 10 mM (1.8 g /100ml)
-KOH (1 M) 2,5 ml/100ml
-All above chemicals dissolved in 70 ml dist. Water
-The pH was adjusted to 7.4
-Add dist water to 100 ml and adjust the pH to 7.6
Appendix
___________________________________________________________________
152
-This solution divided into 10.8 g in plastic tubes and frozen stored.
2. Solution M
10,8 g of solution I (Hepes saline stock solution) was filtered with sterile filter
and mixed with dist water to reach a volume of 100 ml.
250 µl gentamycine sulphate (conc. 20mg / ml) was added.
The pH adjusted to 7.4 and the Osmolarity was measured, measuring value
m (should around 295 mOsm/kg)
3. Percoll®-undiluted
Percoll was taken from the refrigerator and the osmolarity was measured,
measuring value p (should around 15 mOsm / kg)
4. Solution 1 + 9
-5.4 g of the solution1 (Hepes saline stock solution) was filtered through in
sterile filter and mixed with 50.9 g from undiluted Percoll®.
-The osmolarity was measured; measuring; value dp (350mOsm/kg)
5. Computation
1.) Oa = 0,1 x (10m + 9p) = 310
2.) R = [Qa – (0,1 x dp)] / (0,9 x dp) = 0,85
3.) Vp = (10m – 295) / [R x (295 – p)] = 11
6. Percoll®. -Working solutions
a) 100 %age Percoll® - working solution:
= (Vp – 9) x 1.13 x 5 g Percoll® + solution 1 + 9
= (Vp – 9) x 5.65 g Percoll® + solution 1 + 9
-Carefully shacked
-The osmolarity should around 295 mOsm/kg
Appendix
___________________________________________________________________
153
b) 45 %age Percoll® - working solution:
-One half (ca. 33 ml) of the 100 % -solution (V1 corresponds) was taken and
with solution M filled to V2
-V2 = V1 x 100 / 45 (Carefully shacked)
c) 90 %age Percoll® - used solution:
-Another half (ca. 33 ml) of the 100 % -solution (V1 corresponds) was taken
and with solution M filled to V3
-V3 = V1 x 100 / 90
-Carefully shacked
-The prepared solutions can be stored at 4°C in the refrigerator and used for
approximately two weeks
9.3.5 Hepes. 0.1 % BSA-buffer for Supra vital staining
- 0.76 g NaCl
- 0.03 g KCl
- 0.252 g Fructose
- 0.15 g CaCl2
- 0.01g MgCl2
- 100 ml Dist. water
-This buffer portioned into 10 ml aliquots and stored at 23oC
9.3.6 Materials for mf-SCSA
9.3.6.1 Natrium citrate buffer 2.9 % (1000 ml)
- 29 g Tri-Natrium-citrat-dihydrat
- 3.36 g EDTA
-1000 ml dist. water
-The buffer stored at the room temp and can be used up to 3-4 weeks
9.3.6.2 Carnoy’s solution, pH 2 (60 ml)
- 20 ml acetic acid DAB mixed in 40 ml Methanol 99.9 %.
Appendix
___________________________________________________________________
154
9.3.6.3 Citric acid-stock solution 0.1 molar (1000 ml)
- 21.01 g citric acid -monohydrate DAB
- 1000 ml dist. water. Stored in the refrigerator and can be used up to 3-4
weeks
9.3.6.4 Di-Natriumhydrogenphosphat, 0.3 molar solution (100 ml)
- 5.34 g Na2HPO4 x H2O
- 100 ml Dist. water.
- The buffer stored in the refrigerator and can be used up to 3-4 weeks
9.3.6.5 Acridin Orange (AO) stock solution (100 ml)
- 0.1 g Acridin Orange (pure substance)
- 100 ml dist. water.
- The solution stored dark bottle in the refrigerator and can be used up to 3-4
week
Appendix
___________________________________________________________________
155
9.4 MATERIALS FOR THE OVIDUKT-EXPLANT-ASSAY
9.4.1 Equipments and instruments
-Co. TBH GmbH, Langenhagen, Hannover
-Co. Aesculap, TBH GmbH, Langenhagen,
Hannover
-Co. Heraeus, Hamburg
-Co. Dell, USA
-Co. SONY, JAPAN
-Co. Mika medica GmbH BildanalyseVers.
2.0, Copyright 1992), Rosenheim
-Co. Heiland, Hamburg
-Co. Zeiss, Jena
-Co.Panasonic, JAPAN
Co: SONY, JAPAN
-Co. Greiner GmbH, Frickenhausen
-Co. Aesculap, TBH GmbH, Langenhagen,
Hannover
-Co. Serva Feinbiochemica, Heidelberg
-Co. Olympus, JAPAN
-Co. Zeiss (475003), Jena
-Co. SONY, JAPAN
-Co. SONY, JAPAN
-Co. Minitüb GmbH, Heidelberg
-Anatomical tweezers
-Artery forceps
-CO2 incubator (CO2-Auto-Zero)
-Computer connected with
-Monitor (Triniton)
-Computer assisted surface area estimation
programme „Aida“
-One used disposable insulin syringes (1 ml)
-Inverted microscope (IM 35) coupled with
Video camera (Kappa, CF 8 / 1)
-Monitor (WV-3M 1400)
-Petri dishes,size (35 x 10 &100 x 15 mm
-Scissors with two-pointed thighs
-Silicon (Silicone Stopcock Grease, Dow
Corning)
-Scale (OT, 0,01mm)
-Stereomicroscope
-Videocassettes (300 min)
-Video recorder (SLV-E 720, VHS)
-Warm plate (HT 200)
Appendix
___________________________________________________________________
156
9.4.2 PBS Medium (LEFEBVRE and SUAREZ, 1996)
- 0.7948 g NaCl
- 0.0199 g KCl
- 0.0047 g MgCl2
- 0.1321 g NaH2PO4
- 0.02 g KH2PO4
- 0.0097 g CaCl2
-100 ml dist. water.
-The saline solution could up to 3 months in refrigerator used after adjust the
pH to 7.4 and the osmolarity (~290 mOsmol / kg)
9.4.3 TALP Medium (PARRISH et al. 1988)
- 0.5844 g NaC
- 0.0231 g KCl
- 0.21 g NaHCO3
- 0.0041 g NaH2PO4
- 0.0222 g CaCl2
- 0.0082 g MgCl2
- 0.242 g Natrium lactate
- 0.11 g Natrium pyruvate (freshly added at the day of experiment)
- 0.2385 g Hepes (freshly added at the day of experiment)
- 0.6 g BSA (Cohn.s Fraktion V, freshly added at the day of experiment)
- 0.005 g Gentamycin (freshly added at the day of experiment)
- The pH of the prepared solution should be adjusted to 7.54 and the
osmolarity should be between (~280-290 mOsm / kg).
- At the day of the experiment, the working medium should be at least
incubated for one hour in the CO2 incubator (39 oC and 5 % CO2)
Appendix
___________________________________________________________________
157
9.5 MATERIALS FOR IVF
9.5.1 PBS stock solution (1000 ml)
- 3.6 g Natrium pyruvate
- 5.0 g Streptomycin sulphate
- 100.0 g Glucose
- 13.3 g CaCl2 2H2O
- 58.8 g Penicillin (Sodium)
- 1000 ml dist. water
9.5.2 Slice medium (500 ml)
- 500 ml PBS
- 0.5 g BSA
- 0.0056 g Heparin
9.5.3 TCM-air-Medium (100 ml)
- 1.510 g TCM 199
- 0.005 g Gentamycin
- 0.035 g NaHCO3 (extra solved)
- 0.0022 g Natrium pyruvate
- 0.100 g BSA (added after adjustment of the pH to 7.2)
- 100 ml dist. water
- Sterile filtration
The medium stored in the refrigerator and can be used up to 2 weeks
9.5.4 TCM-pure Medium + BSA (Wash drops)
- 1.1510 g TCM 199
- 0.005 g Gentamycin
- 0.220 g NaHCO3 (extra solved)
- 0.0022 g Natrium pyruvate
Appendix
___________________________________________________________________
158
- 100 ml dist. water
- Stirred in open glass for about one hour till the pH reached to7.4
- Sterile filtration. Stored in refrigerator and can be used up to one week
- 0.1 g BSA (freshly added)
9.5.5 TCM-pure Medium+BSA+Suigonan® (Maturation drops)
- 975 µl TCMpur + BSA
- 25 µl Aliquot Suigonan® in 0.9 % NaCl solved (entspricht 10 IU PMSG and 5
IU HCG). Suigonan® (contains 400 IU PMSG and 200 IU HCG, the
drysubstance dissolved in 1 ml 0.9 % NaCl and in divided int 25 µl aliquots
then stored at -20°C for about one month can be used)
9.5.6 sperm-TALP stock solution (500 ml)
- 2.920 g NaCl
- 0.1155 g KCl
- 1.050 g NaHCO3 (extra solved)
- 0.0205 g Na2HPO4.H2O
- 0.147 g CaCl2
- 0.019 g MgCl2
- 0.025 g Gentamycin
- 0.005 g Phenol red
- 2.0175 g Natrium lactate (60 %)
- 1.192 g Hepes (MG 238,3)
- 500 ml Dist. water.
- The pH adjusted to 7.4, sterile filtered and stored in the refrigerator for up to
3 months
9.5.7 fert-TALP stock solution (500 ml)
- 3.329 g NaCl
- 0.1195 g KCl
Appendix
___________________________________________________________________
159
- 1.050 g NaHCO3 (extra solved)
- 0.0205 g Na2HPO4.H2O
- 0.147 g CaCl2
- 0.024 g MgCl2
- 0.0015 g Penicillamine
- 0.005 g Phenol red
- 0.930 g Natrium lactate (69 %)
- 500 ml dist. water
The solution ist im Refrigerator drei Monate haltbar
9.5.8 HHE Stock solution (Heparin / Hypotaurin / Epinephrine)
9.5.8.1 250 µM Epinephrine (50 ml)
- 0.165 g Natrium lactate (60 %)
- Na-metabisulphate (Na2S2O5)
- 50 ml dist. Water
- The pH adjusted to 4.0
- 0.0018 g Epinephrine added to 40 ml of the above solution
9.5.8.2 Hypotaurin 1 mM (10 ml)
- 0.0011 g Hypotaurin
- 10 ml Dist. water
9.5.8.3 Heparin 50 IE (10 ml)
- 0.0028 g Heparin, Serva 177000 IE or
- 0.0027 g Heparin, Serva 186000 IE
- 10 ml dist. water
- Sterile filtration
9.5.8.4 Working medium-Stock solution (40 ml)
- 4 ml Epinephrine solution
- 10 ml Hypotaurin solution
Appendix
___________________________________________________________________
160
- 26 ml dist. water
- 40 µl of the Heparin solution mixed with 80 µl of this Stock solution and
divided in Eppendorf cups and stored at –20oC
- The working medium can be used for 3 months
9.5.9 SOF Medium
- 10 ml SOF/Stock A-Medium
- 0.08 g BSA
- 1 Aliquot SOF / Stock B-Medium
- 1 Aliquot SOF / Stock C-Medium
- 1 Aliquot SOF / Stock D-Medium
- 1 Aliquot SOF / Stock E-Medium
- 1 Aliquot SOF / Stock F-Medium
- All mixed together and the pH adjusted to 7.4 and the osmolarity should be
around 270 then sterile filtered
9.5.9.1 SOF / Stock A-Medium (100 ml)
- 0.630 g NaCl
- 0.0537 g KCl
- 0.210 g NaHCO3 (extra solved)
- 0.0163 g KH2PO4
- 0.025 g CaCl2 2H2O (extra solved)
- 0.0048 g MgCl2
- 0.062 g Natrium lactate (60 %)
- 0.027 g Glucose
- 0.001 g Phenol red
- 100 ml Dist. water
- Sterile filtered and stored at 4oC for up to one month
Appendix
___________________________________________________________________
161
9.5.9.2 SOF / Stock B-Medium (1 ml)
- 0,010 g Natrium pyruvate
- 1 ml dist. water
- The medium portioned into 36 µl aliquots
9.5.9.3 SOF / Stock C-Medium (1 ml)
- 0,015 g Glutamine
- 1 ml dist. water
- The medium portioned into 100 µl aliquots
9.5.9.4 SOF / Stock D-Medium (1 ml)
- 0,010 g Gentamycine
- 1 ml Dist. water
- The Medium is being portioned into 50 µl aliquots in Eppendorf cups
- Stock B, C, and D stored deep frozen at –20oC
- The solution is one month usable
9.5.9.5 SOF / Stock E-Medium (100 ml)
- 1 ml Non-essential aa
- 100 ml Dist. water
- The medium portioned into 100 µl aliquots and stored at 4°C and can be
used for up to 3 months
9.5.9.6 SOF / Stock F-Medium (100 ml)
- 2 ml Essential aa
- 100 ml dist. water
- The medium was portioned into 200 µl aliquots and stored at 4°C and can be
used for up to 3 months.
Statutory Declaration
___________________________________________________________________
162
10 STATUTORY DECLARATION
Hereby I explain that I Independently wrote the thesis with the title:
BINDING CAPACITY OF BULL SPERMATOZOA TO OVIDUCTAL EPITHELIUM IN
VITRO AND ITS RELATION TO SPERM CHROMATIN STABILITY, SPERM
VOLUME REGULATION AND FERTILITY
During the preparation the following assistance were taken up:
1. The statistic evaluation of the results made after consultation with and under
guidance of Ms. Dipl. Dipl.-Phys. Dr. A. M. Petrunkina under use of the statistical
program SAS.
2. At Institute for Reproduction Medicine of the Veterinary University Hannover I
accomplished the attempts with the assistance and aids specified in the chapter
"Materials and Methods``
3. The technical consultation was undertaken from Ms Univ. Prof. Dr. rer. nat. Dr.
med. habil. Edda Töpfer-Petersen and Ms. Apl. Prof. Dr. med. vet. Dagmar Waberski
-Institute for Reproduction Medicine of the Veterinary University Hannover.
I made the thesis at the following institutes: 1) Institute for Reproductive Medicine of
the Veterinary University Hannover and 2) Institute for Animal Science, Mariensee,
Federal Agriculture Research Center (FAL).
The thesis was so far not submitted for an examination or a graduation or for a
similar purpose for evaluation.
I insure that I gave the managing data accordingly after best knowledge completely
and to the truth.
Acknowledgement ___________________________________________________________________
163
11 ACKNOWLEDGEMENTS
First and foremost all thanks to ALLAH (GOD) who give us every thing we have and
is the only beneficial and merciful. I wish to express my sincere appreciation to those
who have given their time, efforts and facilities to make this study possible.
I would like to express my deep gratitude to my supervisor, Prof. Dr. Töpfer-Petersen,
Director of Institute for reproductive Medicine / high School of Veterinary Medicine,
HANNOVER / GERMANY, for providing the opportunity to undertake this research
program, for her kindness in allowing me to work in this institute. Her kind
supervision, breadth of knowledge, valuable advice was greatly appreciated. I would
also like to thank her for her suggestions and for her support and encouragement. I
have enjoyed my association with her and found her support extremely beneficial.
I would like to express the deepest thanks and hearty gratitude to Prof. Dr. Waberski
Institute for Reproductive Medicine / high School of veterinary Medicine, HANNOVER
/ GERMANY, for her help in suggesting the subjects of this thesis, her valuable
assistance, continuous encouragement, her faithfully and patiently advising, sincere
guidance and untiring and patient efforts in direction and supervision of this work.
It is pleasure to acknowledge the efforts of Dr. A. M. Petrunkina in achievement the
statistical analysis. I would like also to thank Prof. Dr. Weitze Institute for
Reproductive Medicine, HANNOVER / GERMANY, for his kind help and friendliness.
Deep gratitude is expressed to Prof. Dr. Günzel-Apel, Prof. Dr. Meinecke and Prof
Dr. Meinecke-Tillmann. Many thanks to Prof. Dr. Heiner Niemann and PD Dr.
Christine Wrenzycki Institute for Animal Science, Mariensee, Federal Agriculture
Research Center (FAL), GERMANY, for providing the opportunity to undertake the
IVF part of this research program.
It is pleasure to acknowledge the efforts of all staff members at the Institute of
Reproductive Medicine who have contributed in this work, especially Dr. Mahnaz
Ekhlasi-Hundrieser, Christiane Hettel, Christine Kochel and Petra Hasenleder for
their helpful, cooperation and support.
Deep Gratitude is also expressed to my colleagues at the institute of reproductive
medicine especially Inken Löhmer, Dorothee Von Witzendorff, Evrim Sahin and Erik
Acknowledgement ___________________________________________________________________
164
Olaf for their kind cooperation and friendliness. I would like to acknowledge the
Egyptian Government "Ministry of High Education" for the personal financial support
during the period of this work. I would like to thank the artificial insemination centre
NORDRIND GMBH BREMEN / HANNOVER for the financial support to undertake
this study. My deep gratitude is also expressed to the Department of
THERIOGENOLOGY, Faculty of Veterinary Medicine/Cairo University / Beni-Suef /
Egypt for proposing me for this fellowship.
Finally, I would like to thank my family Nadia, Amani, Hagar, Sarah and Maryam and
specially my parents. Thank you my Father and my Mother for your support
throughout all of educational endeavours. I love you very much and hope that I can
repay you someday for everything you have done for me.